RNA INTERFERENCE MEDIATED INHIBITION OF PLATELET DERIVED GROWTH FACTOR (PDGF) AND PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating platelet derived growth factor (PDGF) and/or platelet derived growth factor receptor (PDGFr) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of platelet derived growth factor (PDGF) and/or platelet derived growth factor receptor (PDGFr) gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of platelet derived growth factor (PDGF) and/or platelet derived growth factor receptor (PDGFr) genes, such as PDGF and/or PDGFr.

This application is a continuation of U.S. patent application Ser. No.10/923,270, filed Aug. 20, 2004, which is a continuation-in-part ofInternational Patent Application No. PCT/US03/03473, filed Feb. 5, 2003.The parent application Ser. No. 10/923,270 is also acontinuation-in-part of International Patent Application No.PCT/US04/16390, filed May 24, 2004, which is a continuation-in-part ofU.S. patent application Ser. No. 10/826,966, filed Apr. 16, 2004 (nowabandoned), which is continuation-in-part of U.S. patent applicationSer. No. 10/757,803, filed Jan. 14, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/720,448,filed Nov. 24, 2003 (now abandoned), which is a continuation-in-part ofU.S. patent application Ser. No. 10/693,059, filed Oct. 23, 2003, whichis a continuation-in-part of U.S. patent application Ser. No.10/444,853, filed May 23, 2003, which is a continuation-in-part ofInternational Patent Application No. PCT/US03/05346, filed Feb. 20,2003, and a continuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003. The parentapplication Ser. No. 10/923,270 also claims the benefit of U.S.Provisional Application No. 60/543,480, filed Feb. 10, 2004. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing46USCNT”,created on Dec. 12, 2008, which is 182,897 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of platelet derived growthfactor (PDGF) and platelet derived growth factor receptor (PDGFr) geneexpression and/or activity. The present invention is also directed tocompounds, compositions, and methods relating to traits, diseases andconditions that respond to the modulation of expression and/or activityof genes involved in platelet derived growth factor (PDGF) and plateletderived growth factor receptor (PDGFr) gene expression pathways or othercellular processes that mediate the maintenance or development of suchtraits, diseases and conditions. Specifically, the invention relates tosmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable ofmediating RNA interference (RNAi) against platelet derived growth factor(PDGF) and/or platelet derived growth factor receptor (PDGFr), such asPDGF and/or PDGFr gene expression. Such small nucleic acid molecules areuseful, for example, in providing compositions for treatment of traits,diseases and conditions that can respond to modulation of PDGF and/orPDGFr expression in a subject, such as cancer, leukemia, obliterativebronchiolitis, acute glomerulonephritis, stroke (CVA), and/orinflammatory and proliferative traits, diseases, disorders, orconditions.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in Hela cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating platelet derived growth factor (PDGF) and plateletderived growth factor receptor (PDGFr) gene expression using shortinterfering nucleic acid (siNA) molecules. This invention also relatesto compounds, compositions, and methods useful for modulating theexpression and activity of other genes involved in pathways of plateletderived growth factor (PDGF) and platelet derived growth factor receptor(PDGFr) gene expression and/or activity by RNA interference (RNAi) usingsmall nucleic acid molecules. In particular, the instant inventionfeatures small nucleic acid molecules, such as short interfering nucleicacid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression of platelet derived growth factor (PDGF)and/or platelet derived growth factor receptor (PDGFr) genes.

A siNA of the invention can be unmodified or chemically-modified. A siNAof the instant invention can be chemically synthesized, expressed from avector or enzymatically synthesized. The instant invention also featuresvarious chemically-modified synthetic short interfering nucleic acid(siNA) molecules capable of modulating PDGF and/or PDGFr gene expressionor activity in cells by RNA interference (RNAi). The use ofchemically-modified siNA improves various properties of native siNAmolecules through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake. Further, contrary to earlierpublished studies, siNA having multiple chemical modifications retainsits RNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, veterinary,diagnostic, target validation, genomic discovery, genetic engineering,and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofPDGF and/or PDGFr genes encoding proteins, such as proteins comprisingPDGF and/or PDGFr associated with the maintenance and/or development ofcancer, leukemia, obliterative bronchiolitis, acute glomerulonephritis,stroke (CVA), and/or inflammatory and proliferative diseases, traits,conditions and/or disorders, such as genes encoding sequences comprisingthose sequences referred to by GenBank Accession Nos. shown in Table I,referred to herein generally as PDGF and/or PDGFr. The description belowof the various aspects and embodiments of the invention is provided withreference to exemplary PDGF and PDGFr genes referred to herein as PDGFand PDGFr respectively. However, the various aspects and embodiments arealso directed to other PDGF and PDGFr genes, such as homolog genes andtranscript variants, and polymorphisms (e.g., single nucleotidepolymorphism, (SNPs)) associated with certain PDGF and PDGFr genes. Assuch, the various aspects and embodiments are also directed to othergenes that are involved in PDGF and PDGFr mediated pathways of signaltransduction or gene expression that are involved, for example, in themaintenance or development of diseases, traits, or conditions describedherein. These additional genes can be analyzed for target sites usingthe methods described for PDGF and PDGFr genes herein. Thus, themodulation of other genes and the effects of such modulation of theother genes can be performed, determined, and measured as describedherein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGFr gene, wherein said siNA molecule comprises about 15 to about28 base pairs.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGFr gene, wherein said siNA molecule comprises about 15 to about28 base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a PDGFand/or PDGFr RNA via RNA interference (RNAi), wherein the doublestranded siNA molecule comprises a first and a second strand, eachstrand of the siNA molecule is about 15 to about 30 nucleotides inlength, the first strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the PDGF and/or PDGFr RNAfor the siNA molecule to direct cleavage of the PDGF and/or PDGFr RNAvia RNA interference, and the second strand of said siNA moleculecomprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a PDGFand/or PDGFr RNA via RNA interference (RNAi), wherein the doublestranded siNA molecule comprises a first and a second strand, eachstrand of the siNA molecule is about 18 to about 23 nucleotides inlength, the first strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the PDGF and/or PDGFr RNAfor the siNA molecule to direct cleavage of the PDGF and/or PDGFr RNAvia RNA interference, and the second strand of said siNA moleculecomprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a PDGF and/or PDGFr RNA via RNA interference (RNAi),wherein each strand of the siNA molecule is about 18 to about 28nucleotides in length; and one strand of the siNA molecule comprisesnucleotide sequence having sufficient complementarity to the PDGF and/orPDGFr RNA for the siNA molecule to direct cleavage of the PDGF and/orPDGFr RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a PDGF and/or PDGFr RNA via RNA interference (RNAi),wherein each strand of the siNA molecule is about 18 to about 23nucleotides in length; and one strand of the siNA molecule comprisesnucleotide sequence having sufficient complementarity to the PDGF and/orPDGFr RNA for the siNA molecule to direct cleavage of the PDGF and/orPDGFr RNA via RNA interference.

In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a PDGF gene, for example, wherein the PDGFgene comprises PDGF encoding sequence. In one embodiment, the inventionfeatures a siNA molecule that down-regulates expression of a PDGF gene,for example, wherein the PDGF gene comprises PDGF non-coding sequence orregulatory elements involved in PDGF gene expression.

In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a PDGFr gene, for example, wherein thePDGFr gene comprises PDGFr encoding sequence. In one embodiment, theinvention features a siNA molecule that down-regulates expression of aPDGFr gene, for example, wherein the PDGFr gene comprises PDGFrnon-coding sequence or regulatory elements involved in PDGFr geneexpression.

In one embodiment, a siNA of the invention is used to inhibit theexpression of PDGF and/or PDGFr genes or a PDGF and/or PDGFr gene familywherein the genes or gene family sequences share sequence homology. Suchhomologous sequences can be identified as is known in the art, forexample using sequence alignments. siNA molecules can be designed totarget such homologous sequences, for example using perfectlycomplementary sequences or by incorporating non-canonical base pairs,for example mismatches and/or wobble base pairs, that can provideadditional target sequences. In instances where mismatches areidentified, non-canonical base pairs (for example, mismatches and/orwobble bases) can be used to generate siNA molecules that target morethan one gene sequence. In a non-limiting example, non-canonical basepairs such as UU and CC base pairs are used to generate siNA moleculesthat are capable of targeting sequences for differing PDGF and/or PDGFrtargets that share sequence homology. As such, one advantage of usingsiNAs of the invention is that a single siNA can be designed to includenucleic acid sequence that is complementary to the nucleotide sequencethat is conserved between the homologous genes. In this approach, asingle siNA can be used to inhibit expression of more than one geneinstead of using more than one siNA molecule to target the differentgenes.

In one embodiment, the invention features a siNA molecule having RNAiactivity against PDGF RNA, wherein the siNA molecule comprises asequence complementary to any RNA having PDGF encoding sequence, such asthose sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features a siNA molecule having RNAiactivity against PDGF RNA, wherein the siNA molecule comprises asequence complementary to an RNA having variant PDGF encoding sequence,for example other mutant PDGF genes not shown in Table I but known inthe art to be associated with the maintenance and/or development ofcancer, leukemia, obliterative bronchiolitis, acute glomerulonephritis,stroke (CVA), and/or inflammatory and proliferative diseases, traits,conditions and/or disorders. Chemical modifications as shown in TablesIII and IV or otherwise described herein can be applied to any siNAconstruct of the invention. In another embodiment, a siNA molecule ofthe invention includes a nucleotide sequence that can interact withnucleotide sequence of a PDGF gene and thereby mediate silencing of PDGFgene expression, for example, wherein the siNA mediates regulation ofPDGF gene expression by cellular processes that modulate the chromatinstructure or methylation patterns of the PDGF gene and preventtranscription of the PDGF gene.

In one embodiment, the invention features a siNA molecule having RNAiactivity against PDGFr RNA, wherein the siNA molecule comprises asequence complementary to any RNA having PDGFr encoding sequence, suchas those sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features a siNA molecule having RNAiactivity against PDGFr RNA, wherein the siNA molecule comprises asequence complementary to an RNA having variant PDGFr encoding sequence,for example other mutant PDGFr genes not shown in Table I but known inthe art to be associated with the maintenance and/or development ofcancer, leukemia, obliterative bronchiolitis, acute glomerulonephritis,stroke (CVA), and/or inflammatory and proliferative diseases, traits,conditions and/or disorders. Chemical modifications as shown in TablesIII and IV or otherwise described herein can be applied to any siNAconstruct of the invention. In another embodiment, a siNA molecule ofthe invention includes a nucleotide sequence that can interact withnucleotide sequence of a PDGFr gene and thereby mediate silencing ofPDGFr gene expression, for example, wherein the siNA mediates regulationof PDGFr gene expression by cellular processes that modulate thechromatin structure or methylation patterns of the PDGFr gene andprevent transcription of the PDGFr gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of PDGF and/or PDGFr proteins arisingfrom PDGF and/or PDGFr haplotype polymorphisms that are associated witha disease or condition, (e.g., cancer, leukemia, obliterativebronchiolitis, acute glomerulonephritis, stroke (CVA), and/orinflammatory and proliferative traits, diseases, disorders, and/orconditions). Analysis of PDGF and/or PDGFr genes, or PDGF and/or PDGFrprotein or RNA levels can be used to identify subjects with suchpolymorphisms or those subjects who are at risk of developing traits,conditions, or diseases described herein. These subjects are amenable totreatment, for example, treatment with siNA molecules of the inventionand any other composition useful in treating diseases related to PDGFand/or PDGFr gene expression. As such, analysis of PDGF and/or PDGFrprotein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of PDGF and/or PDGFrprotein or RNA levels can be used to predict treatment outcome and todetermine the efficacy of compounds and compositions that modulate thelevel and/or activity of certain PDGF and/or PDGFr proteins associatedwith a trait, condition, or disease.

In one embodiment of the invention a siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a PDGF and/orPDGFr protein. The siNA further comprises a sense strand, wherein saidsense strand comprises a nucleotide sequence of a PDGF and/or PDGFr geneor a portion thereof.

In another embodiment, a siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a PDGF and/or PDGFr protein or a portion thereof. ThesiNA molecule further comprises a sense region, wherein said senseregion comprises a nucleotide sequence of a PDGF and/or PDGFr gene or aportion thereof.

In another embodiment, the invention features a siNA molecule comprisinga nucleotide sequence in the antisense region of the siNA molecule thatis complementary to a nucleotide sequence or portion of sequence of aPDGF and/or PDGFr gene. In another embodiment, the invention features asiNA molecule comprising a region, for example, the antisense region ofthe siNA construct, complementary to a sequence comprising a PDGF and/orPDGFr gene sequence or a portion thereof.

In one embodiment, the antisense region of PDGF and/or PDGFr siNAconstructs comprises a sequence complementary to sequence having any ofSEQ ID NOs. 1-311 or 623-630. In one embodiment, the antisense region ofPDGF and/or PDGFr constructs comprises sequence having any of SEQ IDNOs. 312-622, 639-646, 655-662, 671-678, 687-694, 703-726, 728, 730,732, 735, 737, 739, 741, or 744. In another embodiment, the sense regionof PDGF and/or PDGFr constructs comprises sequence having any of SEQ IDNOs. 1-311, 623-638, 647-654, 663-670, 679-686, 695-702, 727, 729, 731,733, 734, 736, 738, 740, 742, or 743.

In one embodiment, a siNA molecule of the invention comprises any of SEQID NOs. 1-744. The sequences shown in SEQ ID NOs: 1-744 are notlimiting. A siNA molecule of the invention can comprise any contiguousPDGF and/or PDGFr sequence (e.g., about 15 to about 25 or more, or about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous PDGFand/or PDGFr nucleotides).

In yet another embodiment, the invention features a siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a PDGF and/or PDGFr protein, and wherein saidsiNA further comprises a sense strand having about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention a siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a PDGF and/or PDGFr protein, and wherein said siNAfurther comprises a sense region having about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein said sense region and said antisense region arecomprised in a linear molecule where the sense region comprises at leastabout 15 nucleotides that are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a PDGF and/or PDGFr gene.Because PDGF genes (e.g., PDGF superfamily) and PDGFr (e.g., PDGFrsuperfamily) genes can share some degree of sequence homology with eachother, siNA molecules can be designed to target a class of PDGF or PDGFrgenes or alternately specific PDGF or PDGFr genes (e.g., polymorphicvariants) by selecting sequences that are either shared amongstdifferent PDGF or PDGFr targets or alternatively that are unique for aspecific PDGF or PDGFr target. Therefore, in one embodiment, the siNAmolecule can be designed to target conserved regions of PDGF or PDGFrRNA sequences having homology among several PDGF or PDGFr gene variantsso as to target a class of PDGF or PDGFr genes with one siNA molecule.Accordingly, in one embodiment, the siNA molecule of the inventionmodulates the expression of one or both PDGF or PDGFr alleles in asubject. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific PDGF or PDGFr RNAsequence (e.g., a single PDGF or PDGFr allele or PDGF or PDGFr singlenucleotide polymorphism (SNP)) due to the high degree of specificitythat the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for PDGF and/orPDGFr expressing nucleic acid molecules, such as RNA encoding a PDGFand/or PDGFr protein. In one embodiment, the invention features a RNAbased siNA molecule (e.g., a siNA comprising 2′-OH nucleotides) havingspecificity for PDGF and/or PDGFr expressing nucleic acid molecules thatincludes one or more chemical modifications described herein.Non-limiting examples of such chemical modifications include withoutlimitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications, when used invarious siNA constructs, (e.g., RNA based siNA constructs), are shown topreserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds. Furthermore, contraryto the data published by Parrish et al., supra, applicant demonstratesthat multiple (greater than one) phosphorothioate substitutions arewell-tolerated and confer substantial increases in serum stability formodified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a PDGFand/or PDGFr gene. In one embodiment, the double stranded siNA moleculecomprises one or more chemical modifications and each strand of thedouble-stranded siNA is about 21 nucleotides long. In one embodiment,the double-stranded siNA molecule does not contain any ribonucleotides.In another embodiment, the double-stranded siNA molecule comprises oneor more ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule independently comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides, wherein each strand comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides that are complementary to the nucleotides of theother strand. In one embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence or a portion thereof of the PDGFand/or PDGFr gene, and the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence of the PDGF and/or PDGFr gene or a portion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene comprising an antisense region, wherein theantisense region comprises a nucleotide sequence that is complementaryto a nucleotide sequence of the PDGF and/or PDGFr gene or a portionthereof, and a sense region, wherein the sense region comprises anucleotide sequence substantially similar to the nucleotide sequence ofthe PDGF and/or PDGFr gene or a portion thereof. In one embodiment, theantisense region and the sense region independently comprise about 15 toabout 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30) nucleotides, wherein the antisense region comprises about15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotidesof the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by thePDGF and/or PDGFr gene or a portion thereof and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, a siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 32” (Table. IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene, wherein the siNA molecule is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. The sense region can be connected to the antisenseregion via a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene, wherein the siNA molecule comprises about15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNAmolecule comprises one or more chemical modifications. In anotherembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence of a PDGF and/or PDGFr gene or a portion thereof, and thesecond strand of the double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence ora portion thereof of the PDGF and/or PDGFr gene. In another embodiment,one of the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aPDGF and/or PDGFr gene or portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or portion thereof ofthe PDGF and/or PDGFr gene. In another embodiment, each strand of thesiNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and eachstrand comprises at least about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides thatare complementary to the nucleotides of the other strand. The PDGFand/or PDGFr gene can comprise, for example, sequences referred to inTable I.

In one embodiment, a siNA molecule of the invention comprises noribonucleotides. In another embodiment, a siNA molecule of the inventioncomprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a PDGF and/or PDGFr gene or a portionthereof, and the siNA further comprises a sense region comprising anucleotide sequence substantially similar to the nucleotide sequence ofthe PDGF and/or PDGFr gene or a portion thereof. In another embodiment,the antisense region and the sense region each comprise about 15 toabout 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30) nucleotides and the antisense region comprises at leastabout 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30) nucleotides that are complementary tonucleotides of the sense region. The PDGF and/or PDGFr gene cancomprise, for example, sequences referred to in Table I. In anotherembodiment, the siNA is a double stranded nucleic acid molecule, whereeach of the two strands of the siNA molecule independently compriseabout 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40)nucleotides, and where one of the strands of the siNA molecule comprisesat least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 or more) nucleotides that are complementary to the nucleic acidsequence of the PDGF and/or PDGFr gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a PDGF and/or PDGFr gene, or a portion thereof, and thesense region comprises a nucleotide sequence that is complementary tothe antisense region. In one embodiment, the siNA molecule is assembledfrom two separate oligonucleotide fragments, wherein one fragmentcomprises the sense region and the second fragment comprises theantisense region of the siNA molecule. In another embodiment, the senseregion is connected to the antisense region via a linker molecule. Inanother embodiment, the sense region is connected to the antisenseregion via a linker molecule, such as a nucleotide or non-nucleotidelinker. The PDGF and/or PDGFr gene can comprise, for example, sequencesreferred in to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by thePDGF and/or PDGFr gene or a portion thereof and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion, and wherein the siNA molecule has one or more modifiedpyrimidine and/or purine nucleotides. In one embodiment, the pyrimidinenucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In anotherembodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. Inanother embodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In oneembodiment, the pyrimidine nucleotides in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the antisense region are 2′-O-methyl or 2′-deoxy purinenucleotides. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of thesense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene, wherein the siNA molecule is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule, and wherein the fragment comprising the senseregion includes a terminal cap moiety at the 5′-end, the 3′-end, or bothof the 5′ and 3′ ends of the fragment. In one embodiment, the terminalcap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In anotherembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39,or 40) nucleotides. In a non-limiting example, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features a siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, about 15 toabout 40 nucleotides in length. In one embodiment, all pyrimidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidinenucleotides. In one embodiment, the modified nucleotides in the siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe siNA include at least one 2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all uridine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides.

In one embodiment, all guanosine nucleotides present in the siNA are2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further compriseat least one modified internucleotidic linkage, such as phosphorothioatelinkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides arepresent at specifically selected locations in the siNA that aresensitive to cleavage by ribonucleases, such as locations havingpyrimidine nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by thePDGF and/or PDGFr gene or a portion thereof and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion, and wherein the purine nucleotides present in the antisenseregion comprise 2′-deoxy-purine nucleotides. In an alternativeembodiment, the purine nucleotides present in the antisense regioncomprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can comprise a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region.Alternatively, in either of the above embodiments, the antisense regioncan comprise a glyceryl modification at the 3′ end of the antisenseregion. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of theantisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of theinvention comprises sequence complementary to a portion of a PDGF and/orPDGFr transcript having sequence unique to a particular PDGF and/orPDGFr disease related allele, such as sequence comprising a singlenucleotide polymorphism (SNP) associated with the disease specificallele. As such, the antisense region of a siNA molecule of theinvention can comprise sequence complementary to sequences that areunique to a particular allele to provide specificity in mediatingselective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PDGF and/or PDGFr gene, wherein the siNA molecule is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. In another embodiment, the siNA molecule is adouble stranded nucleic acid molecule, where each strand is about 21nucleotides long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region, where about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the PDGF and/or PDGFr gene. In another embodiment, about 21nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the PDGF and/orPDGFr gene. In any of the above embodiments, the 5′-end of the fragmentcomprising said antisense region can optionally include a phosphategroup.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa PDGF and/or PDGFr RNA sequence (e.g., wherein said target RNA sequenceis encoded by a PDGF and/or PDGFr gene involved in the PDGF and/or PDGFrpathway), wherein the siNA molecule does not contain any ribonucleotidesand wherein each strand of the double-stranded siNA molecule is about 15to about 30 nucleotides. In one embodiment, the siNA molecule is 21nucleotides in length. Examples of non-ribonucleotide containing siNAconstructs are combinations of stabilization chemistries shown in TableIV in any combination of Sense/Antisense chemistries, such as Stab 7/8,Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisensestrands or any combination thereof).

In one embodiment, the invention features a chemically synthesizeddouble stranded RNA molecule that directs cleavage of a PDGF and/orPDGFr RNA via RNA interference, wherein each strand of said RNA moleculeis about 15 to about 30 nucleotides in length; one strand of the RNAmolecule comprises nucleotide sequence having sufficient complementarityto the PDGF and/or PDGFr RNA for the RNA molecule to direct cleavage ofthe PDGF and/or PDGFr RNA via RNA interference; and wherein at least onestrand of the RNA molecule optionally comprises one or more chemicallymodified nucleotides described herein, such as without limitationdeoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising a siNAmolecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a PDGF and/or PDGFr gene, whereinthe siNA molecule comprises one or more chemical modifications and eachstrand of the double-stranded siNA is independently about 15 to about 30or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 or more) nucleotides long. In one embodiment, the siNAmolecule of the invention is a double stranded nucleic acid moleculecomprising one or more chemical modifications, where each of the twofragments of the siNA molecule independently comprise about 15 to about40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and whereone of the strands comprises at least 15 nucleotides that arecomplementary to nucleotide sequence of PDGF and/or PDGFr encoding RNAor a portion thereof. In a non-limiting example, each of the twofragments of the siNA molecule comprise about 21 nucleotides. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculecomprising one or more chemical modifications, where each strand isabout 21 nucleotide long and where about 19 nucleotides of each fragmentof the siNA molecule are base-paired to the complementary nucleotides ofthe other fragment of the siNA molecule, wherein at least two 3′terminal nucleotides of each fragment of the siNA molecule are notbase-paired to the nucleotides of the other fragment of the siNAmolecule. In another embodiment, the siNA molecule is a double strandednucleic acid molecule comprising one or more chemical modifications,where each strand is about 19 nucleotide long and where the nucleotidesof each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region and comprising one or more chemical modifications,where about 19 nucleotides of the antisense region are base-paired tothe nucleotide sequence or a portion thereof of the RNA encoded by thePDGF and/or PDGFr gene. In another embodiment, about 21 nucleotides ofthe antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the PDGF and/or PDGFr gene. In anyof the above embodiments, the 5′-end of the fragment comprising saidantisense region can optionally include a phosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a PDGF and/or PDGFr gene,wherein one of the strands of the double-stranded siNA molecule is anantisense strand which comprises nucleotide sequence that iscomplementary to nucleotide sequence of PDGF and/or PDGFr RNA or aportion thereof, the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a PDGF and/or PDGFr gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of PDGF and/or PDGFr RNA or a portion thereof, wherein theother strand is a sense strand which comprises nucleotide sequence thatis complementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a PDGF and/or PDGFr gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of PDGF and/or PDGFr RNA that encodes a protein or portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification. In oneembodiment, each strand of the siNA molecule comprises about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises atleast about 15 nucleotides that are complementary to the nucleotides ofthe other strand. In one embodiment, the siNA molecule is assembled fromtwo oligonucleotide fragments, wherein one fragment comprises thenucleotide sequence of the antisense strand of the siNA molecule and asecond fragment comprises nucleotide sequence of the sense region of thesiNA molecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPDGF and/or PDGFr gene, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, each of the two strands of the siNA molecule can compriseabout 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In oneembodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotidesof each strand of the siNA molecule are base-paired to the complementarynucleotides of the other strand of the siNA molecule. In anotherembodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotidesof each strand of the siNA molecule are base-paired to the complementarynucleotides of the other strand of the siNA molecule, wherein at leasttwo 3′ terminal nucleotides of each strand of the siNA molecule are notbase-paired to the nucleotides of the other strand of the siNA molecule.In another embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as2′-deoxy-thymidine. In one embodiment, each strand of the siNA moleculeis base-paired to the complementary nucleotides of the other strand ofthe siNA molecule. In one embodiment, about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides of the antisense strand are base-paired to the nucleotidesequence of the PDGF and/or PDGFr RNA or a portion thereof. In oneembodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23,24, or 25) nucleotides of the antisense strand are base-paired to thenucleotide sequence of the PDGF and/or PDGFr RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPDGF and/or PDGFr gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of PDGFand/or PDGFr RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification, and wherein the 5′-end of the antisensestrand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPDGF and/or PDGFr gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of PDGFand/or PDGFr RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification, and wherein the nucleotide sequence or aportion thereof of the antisense strand is complementary to a nucleotidesequence of the untranslated region or a portion thereof of the PDGFand/or PDGFr RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPDGF and/or PDGFr gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of PDGFand/or PDGFr RNA or a portion thereof, wherein the other strand is asense strand which comprises nucleotide sequence that is complementaryto a nucleotide sequence of the antisense strand, wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification, and wherein the nucleotide sequence ofthe antisense strand is complementary to a nucleotide sequence of thePDGF and/or PDGFr RNA or a portion thereof that is present in the PDGFand/or PDGFr RNA.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of a siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding PDGF and/orPDGFr and the sense region can comprise sequence complementary to theantisense region. The siNA molecule can comprise two distinct strandshaving complementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides comprising a backbone modified internucleotide linkagehaving Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO₂, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises a 5′-terminal phosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo;

wherein each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W,X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNA,molecule. In another embodiment, the invention features a siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more phosphorothioate internucleotide linkages. Forexample, in a non-limiting example, the invention features achemically-modified short interfering nucleic acid (siNA) having about1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkagesin one siNA strand. In yet another embodiment, the invention features achemically-modified short interfering nucleic acid (siNA) individuallyhaving about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in both siNA strands. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the siNA duplex, for example in the sense strand, theantisense strand, or both strands. The siNA molecules of the inventioncan comprise one or more phosphorothioate internucleotide linkages atthe 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sensestrand, the antisense strand, or both strands. For example, an exemplarysiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orabout one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 10 or more, specificallyabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with orwithout one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore phosphorothioate internucleotide linkages and/or a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, beingpresent in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs, and wherein the siNA can include achemical modification comprising a structure having any of FormulaeI-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3,R8 or R13 serve as points of attachment to the siNA molecule of theinvention.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofa siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against PDGF and/or PDGFr inside acell or reconstituted in vitro system comprising a sense region, whereinone or more pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of thesechemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III andIV herein. In any of these described embodiments, the purine nucleotidespresent in the sense region are alternatively 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides) and one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality of purine nucleotides arepurine ribonucleotides) and any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against PDGF and/or PDGFr inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises a conjugate covalently attached to the chemically-modifiedsiNA molecule. Non-limiting examples of conjugates contemplated by theinvention include conjugates and ligands described in Vargeese et al.,U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by referenceherein in its entirety, including the drawings. In another embodiment,the conjugate is covalently attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.;1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group. In another embodiment, the singlestranded siNA molecule of the invention comprises a 5′-terminalphosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclicphosphate). In another embodiment, the single stranded siNA molecule ofthe invention comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Inyet another embodiment, the single stranded siNA molecule of theinvention comprises one or more chemically modified nucleotides ornon-nucleotides described herein. For example, all the positions withinthe siNA molecule can include chemically-modified nucleotides such asnucleotides having any of Formulae I-VII, or any combination thereof tothe extent that the ability of the siNA molecule to support RNAiactivity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence, wherein one or morepyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.10, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The siNA optionally furthercomprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more)terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, whereinthe terminal nucleotides can further comprise one or more (e.g., 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides). In another embodiment, any modifiednucleotides present in the single stranded siNA molecules of theinvention comprise modified nucleotides having properties orcharacteristics similar to naturally occurring ribonucleotides. Forexample, the invention features siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the single stranded siNA molecules of theinvention are preferably resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemicallymodified nucleotides or non-nucleotides (e.g., having any of FormulaeI-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides)at alternating positions within one or more strands or regions of thesiNA molecule. For example, such chemical modifications can beintroduced at every other position of a RNA based siNA molecule,starting at either the first or second nucleotide from the 3′-end or5′-end of the siNA. In a non-limiting example, a double stranded siNAmolecule of the invention in which each strand of the siNA is 21nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., withcompounds having any of Formulae I-VII, such as such as 2′-deoxy,2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). In another non-limitingexample, a double stranded siNA molecule of the invention in which eachstrand of the siNA is 21 nucleotides in length is featured whereinpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand arechemically modified (e.g., with compounds having any of Formulae I-VII,such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides). Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating theexpression of a PDGF and/or PDGFr gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr gene; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the PDGF and/or PDGFr gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a PDGF and/or PDGFr gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr gene and whereinthe sense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the PDGF and/or PDGFr gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PDGF and/or PDGFr gene within a cellcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr genes; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate the expression of the PDGF and/or PDGFr genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more PDGF and/or PDGFr genes within a cellcomprising: (a) synthesizing one or more siNA molecules of theinvention, which can be chemically-modified, wherein the siNA strandscomprise sequences complementary to RNA of the PDGF and/or PDGFr genesand wherein the sense strand sequences of the siNAs comprise sequencesidentical or substantially similar to the sequences of the target RNAs;and (b) introducing the siNA molecules into a cell under conditionssuitable to modulate the expression of the PDGF and/or PDGFr genes inthe cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PDGF and/or PDGFr gene within a cellcomprising: (a) synthesizing a siNA molecule of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr gene and whereinthe sense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the PDGF and/or PDGFr genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a PDGFand/or PDGFr gene in a tissue explant comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the PDGF and/or PDGFr gene; and (b) introducing the siNA moleculeinto a cell of the tissue explant derived from a particular organismunder conditions suitable to modulate the expression of the PDGF and/orPDGFr gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the PDGF and/or PDGFr gene inthat organism.

In one embodiment, the invention features a method of modulating theexpression of a PDGF and/or PDGFr gene in a tissue explant comprising:(a) synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr gene and whereinthe sense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the PDGF and/or PDGFr gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate the expression ofthe PDGF and/or PDGFr gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PDGF and/or PDGFr gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr genes; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the PDGF and/or PDGFr genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate the expression ofthe PDGF and/or PDGFr genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a PDGF and/or PDGFr gene in a subject or organismcomprising: (a) synthesizing a siNA molecule of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PDGF and/or PDGFr gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in the subject or organism. The level of PDGF and/or PDGFr proteinor RNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one PDGF and/or PDGFr gene in a subject ororganism comprising: (a) synthesizing siNA molecules of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the PDGF and/or PDGFrgenes; and (b) introducing the siNA molecules into the subject ororganism under conditions suitable to modulate the expression of thePDGF and/or PDGFr genes in the subject or organism. The level of PDGFand/or PDGFr protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a PDGF and/or PDGFr gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the PDGF and/or PDGFr gene;and (b) introducing the siNA molecule into a cell under conditionssuitable to modulate the expression of the PDGF and/or PDGFr gene in thecell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PDGF and/or PDGFr gene within a cellcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the PDGF and/or PDGFr gene;and (b) contacting the cell in vitro or in vivo with the siNA moleculeunder conditions suitable to modulate the expression of the PDGF and/orPDGFr genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a PDGF and/or PDGFr gene in a tissue explant comprising:(a) synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the PDGF and/or PDGFr gene;and (b) contacting a cell of the tissue explant derived from aparticular subject or organism with the siNA molecule under conditionssuitable to modulate the expression of the PDGF and/or PDGFr gene in thetissue explant. In another embodiment, the method further comprisesintroducing the tissue explant back into the subject or organism thetissue was derived from or into another subject or organism underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in that subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PDGF and/or PDGFr gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the PDGF and/or PDGFr gene;and (b) introducing the siNA molecules into a cell of the tissue explantderived from a particular subject or organism under conditions suitableto modulate the expression of the PDGF and/or PDGFr genes in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the subject or organism the tissue wasderived from or into another subject or organism under conditionssuitable to modulate the expression of the PDGF and/or PDGFr genes inthat subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a PDGF and/or PDGFr gene in a subject or organismcomprising: (a) synthesizing a siNA molecule of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the PDGF and/or PDGFr gene;and (b) introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PDGF and/or PDGFr gene in a subject ororganism comprising: (a) synthesizing siNA molecules of the invention,which can be chemically-modified, wherein the siNA comprises a singlestranded sequence having complementarity to RNA of the PDGF and/or PDGFrgene; and (b) introducing the siNA molecules into the subject ororganism under conditions suitable to modulate the expression of thePDGF and/or PDGFr genes in the subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a PDGF and/or PDGFr gene in a subject or organismcomprising contacting the subject or organism with a siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe PDGF and/or PDGFr gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing leukemia in a subject or organism comprising contacting thesubject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing obliterative bronchiolitis in a subject or organismcomprising contacting the subject or organism with a siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe PDGF and/or PDGFr gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing acute glomerulonephritis in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate the expression of the PDGF and/orPDGFr gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a stroke (CVA) in a subject or organism comprising contactingthe subject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the PDGF and/or PDGFrgene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an inflammatory disease, disorder, and/or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the PDGF and/or PDGFr gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease, disorder, and/or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the PDGF and/or PDGFr gene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PDGF and/or PDGFr genes in a subject ororganism comprising contacting the subject or organism with one or moresiNA molecules of the invention under conditions suitable to modulatethe expression of the PDGF and/or PDGFr genes in the subject ororganism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., PDGF and/or PDGFr) gene expression through RNAitargeting of a variety of RNA molecules. In one embodiment, the siNAmolecules of the invention are used to target various RNAs correspondingto a target gene. Non-limiting examples of such RNAs include messengerRNA (mRNA), alternate RNA splice variants of target gene(s),post-transcriptionally modified RNA of target gene(s), pre-mRNA oftarget gene(s), and/or RNA templates. If alternate splicing produces afamily of transcripts that are distinguished by usage of appropriateexons, the instant invention can be used to inhibit gene expressionthrough the appropriate exons to specifically inhibit or to distinguishamong the functions of gene family members. For example, a protein thatcontains an alternatively spliced transmembrane domain can be expressedin both membrane bound and secreted forms. Use of the invention totarget the exon containing the transmembrane domain can be used todetermine the functional consequences of pharmaceutical targeting ofmembrane bound as opposed to the secreted form of the protein.Non-limiting examples of applications of the invention relating totargeting these RNA molecules include therapeutic pharmaceuticalapplications, pharmaceutical discovery applications, moleculardiagnostic and gene function applications, and gene mapping, for exampleusing single nucleotide polymorphism mapping with siNA molecules of theinvention. Such applications can be implemented using known genesequences or from partial sequences available from an expressed sequencetag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as PDGF and/or PDGFr family genes. As such, siNA moleculestargeting multiple PDGF and/or PDGFr targets can provide increasedtherapeutic effect. In addition, siNA can be used to characterizepathways of gene function in a variety of applications. For example, thepresent invention can be used to inhibit the activity of target gene(s)in a pathway to determine the function of uncharacterized gene(s) ingene function analysis, mRNA function analysis, or translationalanalysis. The invention can be used to determine potential target genepathways involved in various diseases and conditions towardpharmaceutical development. The invention can be used to understandpathways of gene expression involved in, for example cancer, leukemia,obliterative bronchiolitis, acute glomerulonephritis, stroke (CVA),and/or inflammatory and proliferative traits, diseases, disorders,and/or conditions.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession, for example, PDGF and/or PDGFr genes encodingRNA sequence(s) referred to herein by Genbank Accession number, forexample, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(eg. for a siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target PDGF and/or PDGFr RNAsequence. In another embodiment, the siNA molecules of (a) have strandsof a fixed length, for example about 23 nucleotides in length. In yetanother embodiment, the siNA molecules of (a) are of differing length,for example having strands of about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesin length. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described in Example 6 herein. In anotherembodiment, the assay can comprise a cell culture system in which targetRNA is expressed. In another embodiment, fragments of PDGF and/or PDGFrRNA are analyzed for detectable levels of cleavage, for example, by gelelectrophoresis, northern blot analysis, or RNAse protection assays, todetermine the most suitable target site(s) within the target PDGF and/orPDGFr RNA sequence. The target PDGF and/or PDGFr RNA sequence can beobtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of target RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target RNA sequence. The targetRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for treating orpreventing cancer, leukemia, obliterative bronchiolitis, acuteglomerulonephritis, stroke (CVA), and/or inflammatory and proliferativediseases, traits, conditions and/or disorders in a subject or organismcomprising administering to the subject a composition of the inventionunder conditions suitable for the treatment or prevention of cancer,leukemia, obliterative bronchiolitis, acute glomerulonephritis, stroke(CVA), and/or inflammatory and proliferative diseases, traits,conditions and/or disorders in the subject or organism.

In another embodiment, the invention features a method for validating aPDGF and/or PDGFr gene target, comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein oneof the siNA strands includes a sequence complementary to RNA of a PDGFand/or PDGFr target gene; (b) introducing the siNA molecule into a cell,tissue, subject, or organism under conditions suitable for modulatingexpression of the PDGF and/or PDGFr target gene in the cell, tissue,subject, or organism; and (c) determining the function of the gene byassaying for any phenotypic change in the cell, tissue, subject, ororganism.

In another embodiment, the invention features a method for validating aPDGF and/or PDGFr target comprising: (a) synthesizing a siNA molecule ofthe invention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a PDGF and/or PDGFrtarget gene; (b) introducing the siNA molecule into a biological systemunder conditions suitable for modulating expression of the PDGF and/orPDGFr target gene in the biological system; and (c) determining thefunction of the gene by assaying for any phenotypic change in thebiological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a PDGF and/or PDGFr target gene in abiological system, including, for example, in a cell, tissue, subject,or organism. In another embodiment, the invention features a kitcontaining more than one siNA molecule of the invention, which can bechemically-modified, that can be used to modulate the expression of morethan one PDGF and/or PDGFr target gene in a biological system,including, for example, in a cell, tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing a siNA molecule of the inventionis a mammalian cell. In yet another embodiment, the cell containing asiNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizinga siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications, for example, one or more chemicalmodifications having any of Formulae I-VII or any combination thereofthat increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., have attenuatedor no immunostimulatory properties) comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table IV) or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table IV) or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

By “improved toxicologic profile”, is meant that the chemically modifiedsiNA construct exhibits decreased toxicity in a cell, subject, ororganism compared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In a non-limiting example, siNA molecules withimproved toxicologic profiles are associated with a decreased orattenuated immunostimulatory response in a cell, subject, or organismcompared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In one embodiment, a siNA molecule with an improvedtoxicological profile comprises no ribonucleotides. In one embodiment, asiNA molecule with an improved toxicological profile comprises less than5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In oneembodiment, a siNA molecule with an improved toxicological profilecomprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17,Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27,Stab 28, Stab 29, Stab 30, Stab 31, Stab 32 or any combination thereof(see Table IV). In one embodiment, the level of immunostimulatoryresponse associated with a given siNA molecule can be measured as isknown in the art, for example by determining the level of PKR/interferonresponse, proliferation, B-cell activation, and/or cytokine productionin assays to quantitate the immunostimulatory response of particularsiNA molecules (see, for example, Leifer et al., 2003, J. Immunother.26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety byreference).

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that modulates thebinding affinity between the sense and antisense strands of the siNAconstruct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoa siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that modulates thebinding affinity between the antisense strand of the siNA construct anda complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that modulates thebinding affinity between the antisense strand of the siNA construct anda complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that modulate thepolymerase activity of a cellular polymerase capable of generatingadditional endogenous siNA molecules having sequence homology to thechemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against PDGF and/or PDGFr in a cell,wherein the chemical modifications do not significantly effect theinteraction of siNA with a target RNA molecule, DNA molecule and/orproteins or other factors that are essential for RNAi in a manner thatwould decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against PDGF and/or PDGFrcomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against PDGFand/or PDGFr target RNA comprising (a) introducing nucleotides havingany of Formula I-VII or any combination thereof into a siNA molecule,and (b) assaying the siNA molecule of step (a) under conditions suitablefor isolating siNA molecules having improved RNAi activity against thetarget RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against PDGFand/or PDGFr target DNA comprising (a) introducing nucleotides havingany of Formula I-VII or any combination thereof into a siNA molecule,and (b) assaying the siNA molecule of step (a) under conditions suitablefor isolating siNA molecules having improved RNAi activity against thetarget DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that modulates thecellular uptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against PDGF and/or PDGFr with improved cellular uptakecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against PDGF and/or PDGFr, wherein the siNA construct comprises oneor more chemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, a siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of a siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table IV)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b).

In one embodiment, the method further comprises re-screening thechemically modified siNA molecules of step (c) under conditions suitablefor isolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 to about 25 or morenucleotides of the siNA molecule are complementary to the target nucleicacid or a portion thereof). Alternatively, the siNA is assembled from asingle oligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, a siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctionalsiNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No.60/543,480 filed Feb. 10, 2004 and International PCT Application No.US04/16390, filed May 24, 2004). The multifunctional siNA of theinvention can comprise sequence targeting, for example, two regions ofPDGF and/or PDGFr RNA (see for example target sequences in Tables II andIII).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nuclear RNA (snRNA), ribosomal RNA(rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-codingRNAs can serve as target nucleic acid molecules for siNA mediated RNAinterference in modulating the activity of fRNA or ncRNA involved infunctional or regulatory cellular processes. Aberrant fRNA or ncRNAactivity leading to disease can therefore be modulated by siNA moleculesof the invention. siNA molecules targeting fRNA and ncRNA can also beused to manipulate or alter the genotype or phenotype of a subject,organism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts. For a review, see for example Snyderand Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “PDGF” as used herein is meant any platelet derived growth factorprotein, peptide, or polypeptide having any platelet derived growthfactor activity, such as encoded by PDGF Genbank Accession Nos. shown inTable I. The term PDGF also refers to nucleic acid sequences encodingany platelet derived growth factor protein, peptide, or polypeptidehaving platelet derived growth factor activity. The term “PDGF” is alsomeant to include other platelet derived growth factor encoding sequence,such as other PDGF isoforms, mutant PDGF genes, splice variants of PDGFgenes, and PDGF gene polymorphisms.

By “PDGFr” as used herein is meant any platelet derived growth factorreceptor protein, peptide, or polypeptide having any platelet derivedgrowth factor receptor activity, such as encoded by PDGFr GenbankAccession Nos. shown in Table I. The term PDGFr also refers to nucleicacid sequences encoding any platelet derived growth factor receptorprotein, peptide, or polypeptide having PDGFr activity. The term “PDGFr”is also meant to include other platelet derived growth factor receptorencoding sequence, such as other PDGFr isoforms, mutant PDGFr genes,splice variants of PDGFr genes, and PDGFr gene polymorphisms.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, a siNA molecule ofthe invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate orreduce PDGF and/or PDGFr gene expression are used for preventing ortreating cancer, leukemia, obliterative bronchiolitis, acuteglomerulonephritis, stroke (CVA), and/or inflammatory and proliferativediseases, traits, conditions and/or disorders in a subject or organism.

In one embodiment, the siNA molecules of the invention are used to treatcancer, leukemia, obliterative bronchiolitis, acute glomerulonephritis,stroke (CVA), and/or inflammatory and proliferative diseases, traits,conditions and/or disorders in a subject or organism.

By “proliferative disease” or “cancer” as used herein is meant, anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “leukemia” as used herein is meant any disease, disorder, condition,trait, genotype or phenotype characterized by, for example, theoverproduction of immature atypical leukocytes, such as acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL), and chronic lympocytic leukemia, and anyother leukemia that can respond to the modulation of disease relatedgene expression in a cell or tissue, alone or in combination with othertherapies.

By “inflammatory disease” or “inflammatory condition” as used herein ismeant any disease, condition, trait, genotype or phenotype characterizedby an inflammatory or allergic process as is known in the art, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopicdermatitis, septic shock, rheumatoid arthritis, inflammatory bowldisease, inflammatory pelvic disease, pain, ocular inflammatory disease,celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconioses, and any otherinflammatory disease, condition, trait, genotype or phenotype that canrespond to the modulation of disease related gene expression in a cellor tissue, alone or in combination with other therapies.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples ofsuch nucleic acid molecules consist essentially of sequences defined inthese tables and figures. Furthermore, the chemically modifiedconstructs described in Table IV can be applied to any siNA sequence ofthe invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating cancer, leukemia, obliterative bronchiolitis,acute glomerulonephritis, stroke (CVA), and/or inflammatory andproliferative diseases, traits, conditions and/or disorders in a subjector organism.

For example, the siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or cancer, leukemia, obliterativebronchiolitis, acute glomerulonephritis, stroke (CVA), and/orinflammatory and proliferative diseases, traits, conditions and/ordisorders in a subject or organism. For example, the described moleculescould be used in combination with one or more known compounds,treatments, or procedures to prevent or treat cancer, leukemia,obliterative bronchiolitis, acute glomerulonephritis, stroke (CVA),and/or inflammatory and proliferative diseases, traits, conditionsand/or disorders in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of a siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms a siNA molecule. Non-limiting examplesof such expression vectors are described in Paul et al., 2002, NatureBiotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology,19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina etal., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for a siNA molecule having complementarity to a RNAmolecule referred to by a Genbank Accession numbers, for example GenbankAccession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form asiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.The antisense strand of constructs A-F comprise sequence complementaryto any target nucleic acid sequence of the invention. Furthermore, whena glyceryl moiety (L) is present at the 3′-end of the antisense strandfor any construct shown in FIG. 4 A-F, the modified internucleotidelinkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a PDGFr siNA sequence. Such chemicalmodifications can be applied to any PDGF and/or PDGFr sequence and/orPDGF and/or PDGFr polymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined PDGF and/or PDGFr target sequence, wherein thesense region comprises, for example, about 19, 20, 21, or 22 nucleotides(N) in length, which is followed by a loop sequence of defined sequence(X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in a siNA transcript having specificity for a PDGF and/orPDGFr target sequence and having self-complementary sense and antisenseregions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined PDGF and/or PDGFr target sequence, wherein thesense region comprises, for example, about 19, 20, 21, or 22 nucleotides(N) in length, and which is followed by a 3′-restriction site (R2) whichis adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-mofications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portion's of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC to initiate RNA interferencemediated cleavage of its corresponding target. These design parameterscan include destabilization of each end of the siNA construct (see forexample Schwarz et al., 2003, Cell, 115, 199-208). Such destabilizationcan be accomplished for example by using guanosine-cytidine base pairs,alternate base pairs (e.g., wobbles), or destabilizing chemicallymodified nucleotides at terminal nucleotide positions as is known in theart.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC to initiate RNA interference mediatedcleavage of its corresponding target region. These design parameters caninclude destabilization of each end of the siNA construct (see forexample Schwarz et al., 2003, Cell, 115, 199-208). Such destabilizationcan be accomplished for example by using guanosine-cytidine base pairs,alternate base pairs (e.g., wobbles), or destabilizing chemicallymodified nucleotides at terminal nucleotide positions as is known in theart.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride. (ABI); cappingis performed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to prevent ortreat cancer, leukemia, obliterative bronchiolitis, acuteglomerulonephritis, stroke (CVA), and/or inflammatory and proliferativediseases, traits, conditions and/or disorders, and/or any other trait,disease, disorder or condition that is related to or will respond to thelevels of PDGF and/or PDGFr in a cell or tissue, alone or in combinationwith other therapies. For example, a siNA molecule can comprise adelivery vehicle, including liposomes, for administration to a subject,carriers and diluents and their salts, and/or can be present inpharmaceutically acceptable formulations. Methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Beigelman et al., U.S. Pat.No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). Alternatively, thenucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

In another embodiment, the nucleic acid molecules of the invention canalso be formulated or complexed with polyethyleneimine and derivativesthereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, the nucleic acid molecules of the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which can be delivered by means of aninsufflator. In the insufflator, the powder, e.g., a metered dosethereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquified propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885.

In one embodiment, nucleic acid molecules of the invention areadministered to the central nervous system (CNS) or peripheral nervoussystem (PNS). Experiments have demonstrated the efficient in vivo uptakeof nucleic acids by neurons. As an example of local administration ofnucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. AcidDrug Dev., 8, 75, describe a study in which a 15 mer phosphorothioateantisense nucleic acid molecule to c-fos is administered to rats viamicroinjection into the brain. Antisense molecules labeled withtetramethylrhodamine-isothiocyanate (TRITC) or fluoresceinisothiocyanate (FITC) were taken up by exclusively by neurons thirtyminutes post-injection. A diffuse cytoplasmic staining and nuclearstaining was observed in these cells. As an example of systemicadministration of nucleic acid to nerve cells, Epa et al., 2000,Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse studyin which beta-cyclodextrin-adamantane-oligonucleotide conjugates wereused to target the p75 neurotrophin receptor in neuronallydifferentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, BrainResearch, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199;Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, BrainRes. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable todelivery to and uptake by cells in the CNS and/or PNS.

The delivery of nucleic acid molecules of the invention to the CNS isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of asiNA duplex, or a single self-complementary strand that self hybridizesinto a siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siNA molecule, wherein the sequence isoperably linked to the 3′-end of the open reading frame and wherein thesequence is operably linked to the initiation region, the intron, theopen reading frame and the termination region in a manner which allowsexpression and/or delivery of the siNA molecule.

PDGF/PDGFr Biology and Biochemistry

The following discussion is adapted from R&D Systems Mini-Reviews andTech Notes, Cytokine Mini-Reviews, Platelet Derived Growth Factor,Copyright ©2002 R&D Systems. Historically, it has been a goal of tissueculture researchers to identify substances that provide universal growthor maintenance factor characteristics for various cell lines andisolates. Early tissue culture work demonstrated the superiority ofserum over plasma in stimulating the proliferation of fibroblasts invitro. These observations suggested that a factor released fromplatelets during degranulation was probably responsible for thestimulatory activity. Subsequent investigations clearly demonstratedthat a certain factor released from platelets upon clotting was capableof promoting the growth of various types of cells. This factor wassubsequently purified from platelets and given the name platelet-derivedgrowth factor (PDGF). PDGF is now known to be produced by a number ofcell types besides platelets and it has been found to be a mitogen foralmost all mesenchymally-derived cells, such as blood, muscle,bone/cartilage, and connective tissue cells.

Three forms of PDGF have been identified to date. Each form consists ofa 30 kDa homo- or heterodimeric combination of two genetically distinct,but structurally related, polypeptide chains which are designated A andB chains, respectively. Although considerable work has been done on theprimary structure of each of the chains of human PDGF, the process hasbeen complicated by the fact that each is synthesized as a propeptide,that splice variants exist for the A chain, and that C-terminalproteolytic processing apparently occurs for the B chain and possiblythe A chain as well.

The PDGF A chain is the product of a seven exon chromosomal 7 gene thatgives rise to one of two distinct splice variants. The “long” variant, aprepropeptide of 211 amino acid residues, is synthesized with a signalpeptide of 20 amino acid residues, a propeptide sequence of 66 aminoacid residues, and a mature chain of 125 amino acid residues. Incontrast, the “short” 196 amino acid residue variant shows a 20 aminoacid residue signal sequence, a 66 amino acid residue propeptide, and a16-18 kDa, 110 amino acid residue mature form. The difference betweenthe long and short results from alternative exon usage, with theextended form utilizing exon 6 (18 amino acid residues), but not exon 7,and the short form utilizing exon 7 (3 amino acid residues), but notexon 6. The difference between exon 6 utilization and exon 7 utilizationis not, however, limited to length. Within the 18 amino acid residues ofexon 6 lies an approximately 10 amino acid residue sequence that signalscell retention. Failure to remove this carboxyterminal peptide resultsin a failure to release freely circulating PDGF. Retention under thesecircumstances implies binding to either cell-surface glycosaminoglycansor intercellular matrix. The short version contains no retentionsequence and is secreted into the circulatory system. It is presentlyunclear whether any C-terminal processing of A chains occurs, but theshort variant's 110 amino acid residue mature peptide terminates with anarginine residue. This suggests the possibility, as is the case for theB chain, of a carboxypeptidase-mediated C-terminal truncation to 109amino acid residues with equilization of A and B chain lengths fordimerization. No definitive mechanism for C-terminus processing of thelong form of the A chain has been elucidated and it is not presentlyclear if this form is secreted. One potential N-linked glycosylationsite exists in the mature A chain, but not the B chain, and it issuggested to be utilized. Normal cells such as endothelial cell,macrophages, and fibroblasts are known to concurrently express bothtypes of A chain, with the short version being the most abundant.

The PDGF B chain is the product of a six exon gene on chromosome 22. TheB chain gene is known to be identical to the human c-sis gene, thenormal human cell counterpart to the monkey v-sis (simian sarcoma) virusgene. The protein coded for by c-sis is a 27 kDa, 241 amino acid residueprepropeptide with a 20 amino acid residue signal sequence, 61 aminoacid residue propeptide, and a 16 kDa, 160 amino acid residue “mature”polypeptide. C-terminal cleavage of the mature B chain is believed tooccur, resulting in a final mature product of 12 kDa and 109 amino acidresidues. This is proposed to occur in two stages with a trypsin-likecleavage of residues 111 to 160, followed by a carboxypeptidase cleavageof the remaining arginine at residue 110. As with the long form of chainA, a particular retention sequence approximately 10 amino acid residuesin length has also been identified in the B chain C-terminus. Failure toremove this peptide also results in B chain glycosaminoglycan retention.Dimerization of the A and B chains involves two interchain disulfidebonds, and each chain overlaps the other with a 6 or 7 amino acidresidue extension at either end. Within the 103 overlapping amino acidresidues, the two chains exhibit about 50% sequence identity.

Cells known to express PDGF are diverse. Cells that are reported toexpress the A chain protein (both long and short variants) includefibroblasts, endothelial cells, osteoblasts, platelets, vascular smoothmuscle cells, macrophages and Langerhans cells, and fetal fibroblasts.Cells producing B chain protein include fetal fibroblasts, endothelialcells, platelets, macrophages, neurons and breast ductal epithelium. Anumber of cell types have also been shown to express mRNA for the PDGFchains. In particular, A chain mRNA has been found in type I astrocytes,embryonic endodermal respiratory epithelium, renal mesangial cells, andosteoclasts and chrondrocytes, while B chain mRNA has been localized toembryonic endodermal respiratory epithelium, renal mesangial cells andosteoblasts.

As with many growth factors, PDGF is now considered to be a member of alarger family of factors. In addition to PDGF, this family includes thehomodimeric factors VEGF (vascular endothelial growth factor) and PIGF(placental growth factor), VEGF/PIGF heterodimers, and CTGF (connectivetissue growth factor), a PDGF-like factor secreted by human vascularendothelial cells and fibroblasts. Relative to the PDGF isoforms, VEGFshows distant analogy to PDGF-BB while PIGF corresponds to PDGF-AA. CTGFshows little amino acid identity with PDGF A or B, but reacts withanitsera produced against PDGF. Recently, the status of PDGF has beenre-evaluated based on analysis of its 3-dimensional structure. Alongwith NGF, TGF-beta and glycoprotein hormones (human chorionicgonadotrophic), PDGF is now classified as a member of the cysteine-knotgrowth factor superfamily. Each member of this group occurs as a dimerand is characterized by six cysteines which link together to form a“molecular knot”. The existence of this knot is only revealed by 3-Danalysis, making the criteria for admission to this family unique amongsuperfamilies.

An association is known to exist between alpha-2 macroglobulin(alpha-2M) and the B chain-containing PDGF forms, AB and BB. alpha-2M isa circulating 720 kDa homotetrameric glycoprotein produced byhepatocytes, macrophages and astrocytes whose most widely reportedfunction is that of a scavenger of proteases. Although PDGF does notinteract with the region associated with protease entrapment, it doesbind to other alpha-2M sites not influenced by activation. PDGF-BB hasbeen noted to bind to both fast and slow alpha-2M and does soprincipally in a noncovalent manner. Significantly, the binding isreversible, and PDGF dissociation is suggested to occur at either low pHor when equilibrium kinetics favor dissociation, such as might be thecase when PDGF is removed from circulation by binding to its ownreceptors. Functionally, it is not clear what the role is for B chainbinding to alpha-2M. PDGF binding to the slow form seems to result inits storage, as the alpha-2M receptor binding motif(s) are not exposed,and the PDGF-alpha-2M complex simply circulates. On the other hand,binding to fast or activated alpha-2M results in its rapid clearance viaalpha-2M receptors, bringing the PDGF molecule close to its ownreceptors and perhaps facilitating a secondary PDGF-PDGFR interaction.

Two distinct human PDGF receptor transmembrane binding proteins havebeen identified, a 170 kDa, 1066 amino acid residue alpha-receptor(PDGFR alpha) and a 190 kDa, 1074 amino acid residue beta-receptor(PDGFR beta). The two receptor proteins are structurally related andconsist of an extracellular portion containing five immunoglobulin-likedomains, a single transmembrane region, and an intracellular portionwith a protein-tyrosine kinase domain. A functional PDGF receptor isformed when the two chains of a dimeric PDGF molecule each bind one ofthe above receptor molecules, resulting in their approximation,dimerization and activation. Between the two proteins, there is 44%overall sequence identity. Within the extracellular domain, 30% of theamino acid residues are identical. In addition, a 90 kDa soluble form ofPDGFR alpha, consisting of the extracellular segment of thealpha-receptor, has been found in cell culture medium and in humanplasma. The above two transmembrane receptors share characteristics withother growth factor receptors, such as the M-CSF receptor, c-kit, andthe FGF receptor family. High-affinity binding of PDGF involvesdimerization of the receptors, forming either homodimers or heterodimerswith the alpha and beta receptors/chains. Although it appears that eachsubunit of dimeric PDGF binds to one receptor monomer, it is unclear ifthese PDGF subunits need to be covalently linked. Recent evidencesuggests noncovalently linked B chains are able to activate the PDGFR.

PDGFR alpha binds each of the three forms of PDGF dimers with highaffinity. Although PDGFR beta binds both PDGF-BB and PDGF-AB with highaffinity, it has no reported binding to PDGF-AA. The apparenthigh-affinity binding of the AB dimer to the beta-receptor must beinterpreted with caution, however. Although PDGF-AB can bind to mutant3T3 cells displaying only beta-receptors, it requires 100-fold morePDGF-AB to dimerize the beta-receptors and activate the cells than isrequired for cells also displaying alpha-receptors. Cells known toexpress only alpha-receptors include oligodendroglial progenitors, liverendothelial cells and mesothelium, and platelets. Cells expressing onlybeta-receptors include CNS capillary endothelium, neurons and Ito (fatstoring) cells of the liver, plus monocytes/macrophages. Cells showingcoincident expression of alpha and beta receptors include smooth musclecells, fibroblasts, and Schwann cells.

Receptor binding by PDGF is known to activate intracellular tyrosinekinase, leading to autophosphorylation of the cytoplasmic domain of thereceptor as well as phosphorylation of other intracellular substrates.This reaction is described as one in trans, i.e., the two receptormolecules of the receptor dimer phosphorylate each other. Specificsubstrates identified with the beta-receptor include Src, GTPaseActivating Protein (GAP), phospholypase Cg (PLCg) andphosphotidylinositol 3-phosphate. Both PLCg and GAP seem to bind withdifferent affinities to the a- and beta-receptors, suggesting that theparticular response of a cell depends on the type of receptor itexpresses and the type of PDGF dimer to which it is exposed. In additionto the above, a non-tyrosine phosphorylation-associated signaltransduction pathway can also be activated that involves the zinc fingerprotein erg-1 (early growth response gene 1).

Because there are differences between cells relative to the amounts ofalpha- and beta-receptors that they express, and because of thevariability in PDGF isomer binding to receptors, there is a large rangeof possibilities for biological responses by PDGF. This is reflected inat least four experimental systems where different isoforms of PDGFelicit different results. Vascular smooth muscle cells (SMC) andfibroblasts are both known to express both the alpha- andbeta-receptors. In SMC, PDGF-AA initiates cellular hypertrophy(increased protein synthesis), while BB induces hyperplasia (mitosis).In fibroblasts, the BB isoform initiates chemotaxis, while AA inhibitschemotaxis. In dopaminergic neurons, PDGF-AA promotes embryonic neuronfiber development, while BB serves only as a survival or maintenancefactor. Finally, within the developing lung, the BB isoform regulatesthe growth and number of respiratory tubule epithelial cells, while theAA isoform directs the actual formation of branches arising from therespiratory tubules.

In general, PDGF isoforms are potent mitogens for connective tissuecells, including dermal fibroblasts, arterial smooth muscle cells,chondrocytes and some epithelial and endothelial cells. In addition toits activity as a mitogen, PDGF is chemotactic for fibroblasts andsmooth muscle cells, cells which also respond mitogenically to PDGF, andfor neutrophils and mononuclear cells, cells for which PDGF is not amitogen. There is a considerable body of evidence to indicate that PDGFderived from macrophages, acting as a chemotactic and mitogenic agentfor smooth muscle cells, contributes to the myointimal thickening ofarterial walls characteristic of atherosclerosis. Other reportedactivities for PDGF include the stimulation of granule release byneutrophils and monocytes, the facilitation of steroid synthesis byLeydig cells, stimulation of neutrophil phagocytosis, modulation ofthrombospondin expression and secretion, upregulation of ICAM-1 invascular smooth muscle cells, and the transient induction of T cell IL-2secretion, accompanied by a down-regulation of IL-4 and IFN-gammaproduction, which allow clonal expansion of antigen-activated B and Thelper lymphocytes prior to differentiation. PDGF also appears to beubiquitous in neurons throughout the CNS, where it is suggested to playan important role in neuron survival and regeneration, and in mediationof glial cell proliferation, differentiation and migration.

The use of small interfering nucleic acid molecules targeting PDGF andits receptors therefore provides a class of novel therapeutic agentsthat can be used in the treatment of cancers, proliferative diseases(e.g., restenosis), inflammatory disease, or any other disease orcondition that responds to modulation of PDGF and PDGFr genes.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak Ig cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H20 followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

-   1. The target sequence is parsed in silico into a list of all    fragments or subsequences of a particular length, for example 23    nucleotide fragments, contained within the target sequence. This    step is typically carried out using a custom Perl script, but    commercial sequence analysis programs such as Oligo, MacVector, or    the GCG Wisconsin Package can be employed as well.-   2. In some instances the siNAs correspond to more than one target    sequence; such would be the case for example in targeting different    transcripts of the same gene, targeting different transcripts of    more than one gene, or for targeting both the human gene and an    animal homolog. In this case, a subsequence list of a particular    length is generated for each of the targets, and then the lists are    compared to find matching sequences in each list. The subsequences    are then ranked according to the number of target sequences that    contain the given subsequence; the goal is to find subsequences that    are present in most or all of the target sequences. Alternately, the    ranking can identify subsequences that are unique to a target    sequence, such as a mutant target sequence. Such an approach would    enable the use of siNA to target specifically the mutant sequence    and not effect the expression of the normal sequence.-   3. In some instances the siNA subsequences are absent in one or more    sequences while present in the desired target sequence; such would    be the case if the siNA targets a gene with a paralogous family    member that is to remain untargeted. As in case 2 above, a    subsequence list of a particular length is generated for each of the    targets, and then the lists are compared to find sequences that are    present in the target gene but are absent in the untargeted paralog.-   4. The ranked siNA subsequences can be further analyzed and ranked    according to GC content. A preference can be given to sites    containing 30-70% GC, with a further preference to sites containing    40-60% GC.-   5. The ranked siNA subsequences can be further analyzed and ranked    according to self-folding and internal hairpins. Weaker internal    folds are preferred; strong hairpin structures are to be avoided.-   6. The ranked siNA subsequences can be further analyzed and ranked    according to whether they have runs of GGG or CCC in the sequence.    GGG (or even more Gs) in either strand can make oligonucleotide    synthesis problematic and can potentially interfere with RNAi    activity, so it is avoided whenever better sequences are available.    CCC is searched in the target strand because that will place GGG in    the antisense strand.-   7. The ranked siNA subsequences can be further analyzed and ranked    according to whether they have the dinucleotide UU (uridine    dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end    of the sequence (to yield 3′ UU on the antisense sequence). These    sequences allow one to design siNA molecules with terminal TT    thymidine dinucleotides.-   8. Four or five target sites are chosen from the ranked list of    subsequences as described above. For example, in subsequences having    23 nucleotides, the right 21 nucleotides of each chosen 23-mer    subsequence are then designed and synthesized for the upper (sense)    strand of the siNA duplex, while the reverse complement of the left    21 nucleotides of each chosen 23-mer subsequence are then designed    and synthesized for the lower (antisense) strand of the siNA duplex    (see Tables II and III). If terminal TT residues are desired for the    sequence (as described in paragraph 7), then the two 3′ terminal    nucleotides of both the sense and antisense strands are replaced by    TT prior to synthesizing the oligos.-   9. The siNA molecules are screened in an in vitro, cell culture or    animal model system to identify the most active siNA molecule or the    most preferred target site within the target RNA sequence.-   10. Other design considerations can be used when selecting target    nucleic acid sequences, see, for example, Reynolds et al., 2004,    Nature Biotechnology Advanced Online Publication, 1 Feb. 2004,    doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research,    32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a PDGFand/or PDGFr target sequence is used to screen for target sites in cellsexpressing PDGF and/or PDGFr RNA, such as such human aortic smoothmuscle cells (e.g., HASMC), HeLa cells, or A549 cells. The generalstrategy used in this approach is shown in FIG. 9. A non-limitingexample of such is a pool comprising sequences having any of SEQ ID NOS1-744. Cells expressing PDGF and/or PDGFr (e.g., HASMC, HeLa cells, orA549 cells) are transfected with the pool of siNA constructs and cellsthat demonstrate a phenotype associated with PDGF and/or PDGFrinhibition are sorted. The pool of siNA constructs can be expressed fromtranscription cassettes inserted into appropriate vectors (see forexample FIG. 7 and FIG. 8). The siNA from cells demonstrating a positivephenotypic change (e.g., decreased proliferation, decreased PDGF and/orPDGFr mRNA levels or decreased PDGF and/or PDGFr protein expression),are sequenced to determine the most suitable target site(s) within thetarget PDGF and/or PDGFr RNA sequence.

Example 4 PDGF and/or PDGFr Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the PDGF and/orPDGFr RNA target and optionally prioritizing the target sites on thebasis of folding (structure of any given sequence analyzed to determinesiNA accessibility to the target), by using a library of siNA moleculesas described in Example 3, or alternately by using an in vitro siNAsystem as described in Example 6 herein. siNA molecules were designedthat could bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting PDGF and/or PDGFr RNA targets. Theassay comprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with PDGF and/or PDGFr target RNA. A Drosophila extractderived from syncytial blastoderm is used to reconstitute RNAi activityin vitro. Target RNA is generated via in vitro transcription from anappropriate PDGF and/or PDGFr expressing plasmid using T7 RNA polymeraseor via chemical synthesis as described herein. Sense and antisense siNAstrands (for example 20 uM each) are annealed by incubation in buffer(such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mMmagnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C.,then diluted in lysis buffer (for example 100 mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can bemonitored by gel electrophoresis on an agarose gel in TBE buffer andstained with ethidium bromide. The Drosophila lysate is prepared usingzero to two-hour-old embryos from Oregon R flies collected on yeastedmolasses agar that are dechorionated and lysed. The lysate iscentrifuged and the supernatant isolated. The assay comprises a reactionmixture containing 50% lysate [vol/vol], RNA (10-50 μM finalconcentration), and 10% [vol/vol] lysis buffer containing siNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer. (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in thePDGF and/or PDGFr RNA target for siNA mediated RNAi cleavage, wherein aplurality of siNA constructs are screened for RNAi mediated cleavage ofthe PDGF and/or PDGFr RNA target, for example, by analyzing the assayreaction by electrophoresis of labeled target RNA, or by northernblotting, as well as by other methodology well known in the art.

Example 7 Nucleic Acid Inhibition of PDGF and/or PDGFr Target RNA

siNA molecules targeted to the human PDGF and/or PDGFr RNA are designedand synthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within thePDGF and/or PDGFr RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting PDGF and/orPDGFr. First, the reagents are tested in cell culture using, forexample, HASMC, HeLa cells, or A549 cells to determine the extent of RNAand protein inhibition. siNA reagents (e.g.; see Tables II and III) areselected against the PDGF and/or PDGFr target as described herein. RNAinhibition is measured after delivery of these reagents by a suitabletransfection agent to, for example, HASMC, HeLa cells, or A549 cells.Relative amounts of target RNA are measured versus actin using real-timePCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparisonis made to a mixture of oligonucleotide sequences made to unrelatedtargets or to a randomized siNA control with the same overall length andchemistry, but randomly substituted at each position. Primary andsecondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siNA to Cells

Cells such as HASMC, HeLa cells, or A549 cells are seeded, for example,at 1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) theday before transfection. siNA (final concentration, for example 20 nM)and cationic lipid (e.g., final concentration 2 μg/ml) are complexed inEGM basal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrenetubes. Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, IOU RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcycler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of PDGF and/orPDGFr Gene Expression Cell Culture

There are numerous cell culture systems that can be used to analyzereduction of PDGF and/or PDGFr levels either directly or indirectly bymeasuring downstream effects. For example, HASMC, HeLa, or A549 cellscan be used in cell culture experiments to assess the efficacy ofnucleic acid molecules of the invention. As such, HASMC, HeLa, or A549cells treated with nucleic acid molecules of the invention (e.g., siNA)targeting PDGF and/or PDGFr RNA would be expected to have decreased PDGFand/or PDGFr expression capacity following stimulation withpro-inflammatory cytokines compared to matched control nucleic acidmolecules having a scrambled or inactive sequence. In a non-limitingexample, HASMC, HeLa, or A549 cells are cultured and PDGF and/or PDGFrexpression is quantified, for example by time-resolvedimmunofluorometric assay. PDGF and/or PDGFr messenger-RNA expression isquantitated with RT-PCR in cultured cells. Untreated cells are comparedto cells treated with siNA molecules transfected with a suitablereagent, for example, a cationic lipid such as lipofectamine, and PDGFand/or PDGFr protein and RNA levels are quantitated. Dose responseassays are then performed to establish dose dependent inhibition of PDGFand/or PDGFr expression.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-PDGF and/or PDGFr agents in animalmodels is an important prerequisite to human clinical trials. Barisoniet al., 1995, Am J. Pathol., 147, 1728-35 describe a transgenic mousemodel of polycystic kidney disease. Adult polycystic kidney disease isbelieved to be the most frequent ( 1/500) inherited genetic disorder inhumans. Barisoni et al., supra generated a genetic model of the diseasein transgenic mice by introducing a deregulated proto-oncogene c-mycspecifically expressed in the kidney. All transgenic lines produceddevelop adult polycystic kidney disease in a reproducible manner. Theclinical phenotype observed in mice is present at birth and leads torenal insufficiency in adulthood. Barisoni et al., supra determined thatabnormal proliferation and programmed cell death are responsible forcystogenesis in polycystic kidney disease. Furthermore, this phenomenais controlled by a specific c-myc mechanism independent of the p53pathway. A similar mechanism also prevails in human autosomal dominantpolycystic kidney disease. Therefore, this murine model provides auseful model to understand the polycystic kidney disease pathogenesisand can be used to evaluate potential therapeutic agents such as siNAmolecules of the invention.

Other animal models known in the art can be used to evaluate siNAmolecules of the invention targeting PDGF and PDGFr for other diseaseconditions, see for example Karas et al., 1992, Coronary intimalproliferation after balloon injury and stenting in swine: An animalmodel of restenosis. J Am Coll Cardiol. 20, 467-474; Hele, 2001, Theheterotopic tracheal allograft as an animal model of obliterativebronchiolitis. Respir. Res., 2, 169-183; Floege et al. 1999, Am. J.Pathol., 154, 169 (animal model of acute glomerulonephritis). Similarly,using various animal models of oncology known in the art, animalstreated with siNA molecules of the invention targeting PDGF and/or PDGFrRNA can be evaluated for clinical response (e.g., decreased tumorsize/metastasis) and/or decreased levels of Myc RNA or protein.

Example 9 RNAi Mediated Inhibition of PDGF and/or PDGFr Expression

siNA constructs (Table III) are tested for efficacy in reducing PDGFand/or PDGFr RNA expression in, for example, HASMC, HeLa cells, or A549cells. Cells are plated approximately 24 hours before transfection in96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at thetime of transfection cells are 70-90% confluent. For transfection,annealed siNAs are mixed with the transfection reagent (Lipofectamine2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutesat room temperature. The siNA transfection mixtures are added to cellsto give a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the target gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data isaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

Example 10 Indications

The present body of knowledge in PDGF and PDGFr research indicates theneed for methods and compounds that can regulate PDGF and PDGFr geneproduct expression for research, diagnostic, and therapeutic use. Asdescribed herein, the nucleic acid molecules of the present inventioncan be used to treat leukemias, including acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), Acute lymphocytic leukemia(ALL), and chronic lymphocytic leukemia; ovarian cancer, breast cancer,cancers of the head and neck, lymphomas, such as mantle cell lymphoma,non-Hodgkin's lymphoma, and Burkitt's lymphoma, adenoma, squamous cellcarcinoma, laryngeal carcinoma, multiple myeloma, melanoma, colorectalcancer, prostate cancer, and inflammatory and proliferative diseasessuch as restenosis, polycystic kidney disease, obliterativebronchiolitis, acute glomerulonephritis, stroke (CVA), and any otherdiseases or conditions that are related to or will respond to the levelsof PDGF and/or PDGFr in a cell or tissue, alone or in combination withother therapies.

The use of radiation treatments and chemotherapeutics such asGemcytabine and cyclophosphamide are non-limiting examples ofchemotherapeutic agents that can be combined with or used in conjunctionwith the nucleic acid molecules (e.g. siNA molecules) of the instantinvention. Those skilled in the art will recognize that otheranti-cancer and/or antiproliferative compounds and therapies can besimilarly be readily combined with the nucleic acid molecules of theinstant invention (e.g. siNA molecules) and are hence within the scopeof the instant invention. Such compounds and therapies are well known inthe art (see for example Cancer: Principles and Practice of Oncology,Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A.,J.B. Lippincott Company, Philadelphia, USA; incorporated herein byreference) and include, without limitations, folates, antifolates,pyrimidine analogs, fluoropyrimidines, purine analogs, adenosineanalogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids,antibiotics, anthacyclins, platinum analogs, alkylating agents,nitrosoureas, plant derived compounds such as vinca alkaloids,epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiationtherapy, surgery, nutritional supplements, gene therapy, radiotherapy,for example 3D-CRT, immunotoxin therapy, for example ricin, andmonoclonal antibodies. Specific examples of chemotherapeutic compoundsthat can be combined with or used in conjunction with the nucleic acidmolecules of the invention include, but are not limited to, Paclitaxel;Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen;Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin;Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin;Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogenmustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide;4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11,Camptothecin-11, Campto) Tamoxifen, Herceptin; IMC C225; ABX-EGF: andcombinations thereof are non-limiting examples of compounds and/ormethods that can be combined with or used in conjunction with thenucleic acid molecules (e.g. siNA) of the instant invention. Thoseskilled in the art will recognize that other drug compounds andtherapies can be similarly be readily combined with the nucleic acidmolecules of the instant invention (e.g., siNA molecules) are hencewithin the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNAs are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I PDGFr and PDGF Accession Numbers NM_002609 Homo sapiensplatelet-derived growth factor receptor, beta polypeptide (PDGFRB), mRNAgi|15451788|ref|NM_002609.2|[15451788] NM_006206 Homo sapiensplatelet-derived growth factor receptor, alpha polypeptide (PDGFRA),mRNA gi|15451787|ref|NM_006206.2|[15451787] 1: BD166138 Platelet-derivedgrowth factor receptorsgi|27871950|dbj|BD166138.1||pat|JP|2002186490|3[27871950] BD166137Platelet-derived growth factor receptorsgi|27871949|dbj|BD166137.1||pat|JP|2002186490|2[27871949] BD166136Platelet-derived growth factor receptorsgi|27871948|dbj|BD166136.1||pat|JP|2002186490|1[27871948] NM_033016 Homosapiens platelet-derived growth factor beta polypeptide (simian sarcomaviral (v-sis) oncogene homolog) (PDGFB), transcript variant 2, mRNAgi|15451785|ref|NM_033016.1|[15451785] NM_002608 Homo sapiensplatelet-derived growth factor beta polypeptide (simian sarcoma viral(v-sis) oncogene homolog) (PDGFB), transcript variant 1, mRNAgi|4505680|ref|NM_002608.1|[4505680] M59423 Human platelet-derivedgrowth factor A-chain (PDGF) gene, 5′ end and promoter regiongi|189877|gb|M59423.1|HUMPGDF[189877] Y14326 Homo sapiens plateletderived growth factor, B-chain 5′UTRgi|2832416|emb|Y14326.1|HSPDGFBC[2832416] X83705 H. sapiens mRNA forc-sis proto-oncogene gi|951023|emb|X83705.1|HSRNASIS[951023] X00562Human proto-oncogene c-sis fragment for PDGF B chain precursor(platelet-derived growth factor) gi|36477|emb|X00562.1|HSSISB5[36477]X00561 Human proto-oncogene c-sis fragment for PDGF B chain precursor(platelet-derived growth factor) gi|36474|emb|X00561.1|HSSISB4[36474]X00560 Human proto-oncogene c-sis fragment for PDGF B chain precursor(platelet-derived growth factor) gi|36472|emb|X00560.1|HSSISB3[36472]X00559 Human proto-oncogene c-sis fragment for PDGF B chain precursor(platelet-derived growth factor) gi|36470|emb|X00559.1|HSSISB2[36470]X00556 Human proto-oncogene c-sis fragment for PDGF B chain precursor(platelet-derived growth factor) gi|36468|emb|X00556.1|HSSISB1[36468]X02811 Human mRNA for platelet-derived growth factor B chain (PDGF-B)gi|35371|emb|X02811.1|HSPDGFB[35371] X03795 Human mRNA for plateletderived growth factor A-chain (PDGF-A)gi|35365|emb|X03795.1|HSPDGFAR[35365] X06374 Human mRNA forplatelet-derived growth factor PDGF-Agi|35363|emb|X06374.1|HSPDGFA[35363] AF417590 Homo sapiensplatelet-derived growth factor A chain (PDGFA) gene, exon 1 and partialsequence gi|16033732|gb|AF417590.1|AF417590[16033732] NM_006206 Homosapiens platelet-derived growth factor receptor, alpha polypeptide(PDGFRA), mRNA gi|15451787|ref|NM_006206.2|[15451787] AF244813 Homosapiens platelet-derived growth factor C mRNA, complete cdsgi|8886883|gb|AF244813.1|AF244813[8886883] NM_002607 Homo sapiensplatelet-derived growth factor alpha polypeptide (PDGFA), transcriptvariant 1, mRNA gi|15208657|ref|NM_002607.2|[15208657]

TABLE II PDGFRB siNA AND TARGET SEQUENCES PDGFRB NM_002609.2 Seq Seq SeqPos Seq ID UPos Upper seq ID LPos Lower seq ID 3 CCCCUCAGCCCUGCUGCCC 1 3CCCCUCAGCCCUGCUGCCC 1 21 GGGCAGCAGGGCUGAGGGG 312 21 CAGCACGAGCCUGUGCUCG2 21 CAGCACGAGCCUGUGCUCG 2 39 CGAGCACAGGCUCGUGCUG 313 39GCCCUGCCCAACGCAGACA 3 39 GCCCUGCCCAACGCAGACA 3 57 UGUCUGCGUUGGGCAGGGC314 57 AGCCAGACCCAGGGCGGCC 4 57 AGCCAGACCCAGGGCGGCC 4 75GGCCGCCCUGGGUCUGGCU 315 75 CCCUCUGGCGGCUCUGCUC 5 75 CCCUCUGGCGGCUCUGCUC5 93 GAGCAGAGCCGCCAGAGGG 316 93 CCUCCCGAAGGAUGCUUGG 6 93CCUCCCGAAGGAUGCUUGG 6 111 CCAAGCAUCCUUCGGGAGG 317 111GGGAGUGAGGCGAAGCUGG 7 111 GGGAGUGAGGCGAAGCUGG 7 129 CCAGCUUCGCCUCACUCCC318 129 GGCGCUCCUCUCCCCUACA 8 129 GGCGCUCCUCUCCCCUACA 8 147UGUAGGGGAGAGGAGCGCC 319 147 AGCAGCCCCCUUCCUCCAU 9 147AGCAGCCCCCUUCCUCCAU 9 165 AUGGAGGAAGGGGGCUGCU 320 165UCCCUCUGUUCUCCUGAGC 10 165 UCCCUCUGUUCUCCUGAGC 10 183GCUCAGGAGAACAGAGGGA 321 183 CCUUCAGGAGCCUGCACCA 11 183CCUUCAGGAGCCUGCACCA 11 201 UGGUGCAGGCUCCUGAAGG 322 201AGUCCUGCCUGUCCUUCUA 12 201 AGUCCUGCCUGUCCUUCUA 12 219UAGAAGGACAGGCAGGACU 323 219 ACUCAGCUGUUACCCACUC 13 219ACUCAGCUGUUACCCACUC 13 237 GAGUGGGUAACAGCUGAGU 324 237CUGGGACCAGCAGUCUUUC 14 237 CUGGGACCAGCAGUCUUUC 14 255GAAAGACUGCUGGUCCCAG 325 255 CUGAUAACUGGGAGAGGGC 15 255CUGAUAACUGGGAGAGGGC 15 273 GCCCUCUCCCAGUUAUCAG 326 273CAGUAAGGAGGACUUCCUG 16 273 CAGUAAGGAGGACUUCCUG 16 291CAGGAAGUCCUCCUUACUG 327 291 GGAGGGGGUGACUGUCCAG 17 291GGAGGGGGUGACUGUCCAG 17 309 CUGGACAGUCACCCCCUCC 328 309GAGCCUGGAACUGUGCCCA 18 309 GAGCCUGGAACUGUGCCCA 18 327UGGGCACAGUUCCAGGCUC 329 327 ACACCAGAAGCCAUCAGCA 19 327ACACCAGAAGCCAUCAGCA 19 345 UGCUGAUGGCUUCUGGUGU 330 345AGCAAGGACACCAUGCGGC 20 345 AGCAAGGACACCAUGCGGC 20 363GCCGCAUGGUGUCCUUGCU 331 363 CUUCCGGGUGCGAUGCCAG 21 363CUUCCGGGUGCGAUGCCAG 21 381 CUGGCAUCGCACCCGGAAG 332 381GCUCUGGCCCUCAAAGGCG 22 381 GCUCUGGCCCUCAAAGGCG 22 399CGCCUUUGAGGGCCAGAGC 333 399 GAGCUGCUGUUGCUGUCUC 23 399GAGCUGCUGUUGCUGUCUC 23 417 GAGACAGCAACAGCAGCUC 334 417CUCCUGUUACUUCUGGAAC 24 417 CUCCUGUUACUUCUGGAAC 24 435GUUCCAGAAGUAACAGGAG 335 435 CCACAGAUCUCUCAGGGCC 25 435CCACAGAUCUCUCAGGGCC 25 453 GGCCCUGAGAGAUCUGUGG 336 453CUGGUCGUCACACCCCCGG 26 453 CUGGUCGUCACACCCCCGG 26 471CCGGGGGUGUGACGACCAG 337 471 GGGCCAGAGCUUGUCCUCA 27 471GGGCCAGAGCUUGUCCUCA 27 489 UGAGGACAAGCUCUGGCCC 338 489AAUGUCUCCAGCACCUUCG 28 489 AAUGUCUCCAGCACCUUCG 28 507CGAAGGUGCUGGAGACAUU 339 507 GUUCUGACCUGCUCGGGUU 29 507GUUCUGACCUGCUCGGGUU 29 525 AACCCGAGCAGGUCAGAAC 340 525UCAGCUCCGGUGGUGUGGG 30 525 UCAGCUCCGGUGGUGUGGG 30 543CCCACACCACCGGAGCUGA 341 543 GAACGGAUGUCCCAGGAGC 31 543GAACGGAUGUCCCAGGAGC 31 561 GCUCCUGGGACAUCCGUUC 342 561CCCCCACAGGAAAUGGCCA 32 561 CCCCCACAGGAAAUGGCCA 32 579UGGCCAUUUCCUGUGGGGG 343 579 AAGGCCCAGGAUGGCACCU 33 579AAGGCCCAGGAUGGCACCU 33 597 AGGUGCCAUCCUGGGCCUU 344 597UUCUCCAGCGUGCUCACAC 34 597 UUCUCCAGCGUGCUCACAC 34 615GUGUGAGCACGCUGGAGAA 345 615 CUGACCAACCUCACUGGGC 35 615CUGACCAACCUCACUGGGC 35 633 GCCCAGUGAGGUUGGUCAG 346 633CUAGACACGGGAGAAUACU 36 633 CUAGACACGGGAGAAUACU 36 651AGUAUUCUCCCGUGUCUAG 347 651 UUUUGCACCCACAAUGACU 37 651UUUUGCACCCACAAUGACU 37 669 AGUCAUUGUGGGUGCAAAA 348 669UCCCGUGGACUGGAGACCG 38 669 UCCCGUGGACUGGAGACCG 38 687CGGUCUCCAGUCCACGGGA 349 687 GAUGAGCGGAAACGGCUCU 39 687GAUGAGCGGAAACGGCUCU 39 705 AGAGCCGUUUCCGCUCAUC 350 705UACAUCUUUGUGCCAGAUC 40 705 UACAUCUUUGUGCCAGAUC 40 723GAUCUGGCACAAAGAUGUA 351 723 CCCACCGUGGGCUUCCUCC 41 723CCCACCGUGGGCUUCCUCC 41 741 GGAGGAAGCCCACGGUGGG 352 741CCUAAUGAUGCCGAGGAAC 42 741 CCUAAUGAUGCCGAGGAAC 42 759GUUCCUCGGCAUCAUUAGG 353 759 CUAUUCAUCUUUCUCACGG 43 759CUAUUCAUCUUUCUCACGG 43 777 CCGUGAGAAAGAUGAAUAG 354 777GAAAUAACUGAGAUCACCA 44 777 GAAAUAACUGAGAUCACCA 44 795UGGUGAUCUCAGUUAUUUC 355 795 AUUCCAUGCCGAGUAACAG 45 795AUUCCAUGCCGAGUAACAG 45 813 CUGUUACUCGGCAUGGAAU 356 813GACCCACAGCUGGUGGUGA 46 813 GACCCACAGCUGGUGGUGA 46 831UCACCACCAGCUGUGGGUC 357 831 ACACUGCACGAGAAGAAAG 47 831ACACUGCACGAGAAGAAAG 47 849 CUUUCUUCUCGUGCAGUGU 358 849GGGGACGUUGCACUGCCUG 48 849 GGGGACGUUGCACUGCCUG 48 867CAGGCAGUGCAACGUCCCC 359 867 GUCCCCUAUGAUCACCAAC 49 867GUCCCCUAUGAUCACCAAC 49 885 GUUGGUGAUCAUAGGGGAC 360 885CGUGGCUUUUCUGGUAUCU 50 885 CGUGGCUUUUCUGGUAUCU 50 903AGAUACCAGAAAAGCCACG 361 903 UUUGAGGACAGAAGCUACA 51 903UUUGAGGACAGAAGCUACA 51 921 UGUAGCUUCUGUCCUCAAA 362 921AUCUGCAAAACCACCAUUG 52 921 AUCUGCAAAACCACCAUUG 52 939CAAUGGUGGUUUUGCAGAU 363 939 GGGGACAGGGAGGUGGAUU 53 939GGGGACAGGGAGGUGGAUU 53 957 AAUCCACCUCCCUGUCCCC 364 957UCUGAUGCCUACUAUGUCU 54 957 UCUGAUGCCUACUAUGUCU 54 975AGACAUAGUAGGCAUCAGA 365 975 UACAGACUCCAGGUGUCAU 55 975UACAGACUCCAGGUGUCAU 55 993 AUGACACCUGGAGUCUGUA 366 993UCCAUCAACGUCUCUGUGA 56 993 UCCAUCAACGUCUCUGUGA 56 1011UCACAGAGACGUUGAUGGA 367 1011 AACGCAGUGCAGACUGUGG 57 1011AACGCAGUGCAGACUGUGG 57 1029 CCACAGUCUGCACUGCGUU 368 1029GUCCGCCAGGGUGAGAACA 58 1029 GUCCGCCAGGGUGAGAACA 58 1047UGUUCUCACCCUGGCGGAC 369 1047 AUCACCCUCAUGUGCAUUG 59 1047AUCACCCUCAUGUGCAUUG 59 1065 CAAUGCACAUGAGGGUGAU 370 1065GUGAUCGGGAAUGAGGUGG 60 1065 GUGAUCGGGAAUGAGGUGG 60 1083CCACCUCAUUCCCGAUCAC 371 1083 GUCAACUUCGAGUGGACAU 61 1083GUCAACUUCGAGUGGACAU 61 1101 AUGUCCACUCGAAGUUGAC 372 1101UACCCCCGCAAAGAAAGUG 62 1101 UACCCCCGCAAAGAAAGUG 62 1119CACUUUCUUUGCGGGGGUA 373 1119 GGGCGGCUGGUGGAGCCGG 63 1119GGGCGGCUGGUGGAGCCGG 63 1137 CCGGCUCCACCAGCCGCCC 374 1137GUGACUGACUUCCUCUUGG 64 1137 GUGACUGACUUCCUCUUGG 64 1155CCAAGAGGAAGUCAGUCAC 375 1155 GAUAUGCCUUACCACAUCC 65 1155GAUAUGCCUUACCACAUCC 65 1173 GGAUGUGGUAAGGCAUAUC 376 1173CGCUCCAUCCUGCACAUCC 66 1173 CGCUCCAUCCUGCACAUCC 66 1191GGAUGUGCAGGAUGGAGCG 377 1191 CCCAGUGCCGAGUUAGAAG 67 1191CCCAGUGCCGAGUUAGAAG 67 1209 CUUCUAACUCGGCACUGGG 378 1209GACUCGGGGACCUACACCU 68 1209 GACUCGGGGACCUACACCU 68 1227AGGUGUAGGUCCCCCAGUC 379 1227 UGCAAUGUGACGGAGAGUG 69 1227UGCAAUGUGACGGAGAGUG 69 1245 CACUCUCCGUCACAUUGCA 380 1245GUGAAUGACCAUCAGGAUG 70 1245 GUGAAUGACCAUCAGGAUG 70 1263CAUCCUGAUGGUCAUUCAC 381 1263 GAAAAGGCCAUCAACAUCA 71 1263GAAAAGGCCAUCAACAUCA 71 1281 UGAUGUUGAUGGCCUUUUC 382 1281ACCGUGGUUGAGAGCGGCU 72 1281 ACCGUGGUUGAGAGCGGCU 72 1299AGCCGCUCUCAACCACGGU 383 1299 UACGUGCGGCUCCUGGGAG 73 1299UACGUGCGGCUCCUGGGAG 73 1317 CUCCCAGGAGCCGCACGUA 384 1317GAGGUGGGCACACUACAAU 74 1317 GAGGUGGGCACACUACAAU 74 1335AUUGUAGUGUGCCCACCUC 385 1335 UUUGCUGAGCUGCAUCGGA 75 1335UUUGCUGAGCUGCAUCGGA 75 1353 UCCGAUGCAGCUCAGCAAA 386 1353AGCCGGACACUGCAGGUAG 76 1353 AGCCGGACACUGCAGGUAG 76 1371CUACCUGCAGUGUCCGGCU 387 1371 GUGUUCGAGGCCUACCCAC 77 1371GUGUUCGAGGCCUACCCAC 77 1389 GUGGGUAGGCCUCGAACAC 388 1389CCGCCCACUGUCCUGUGGU 78 1389 CCGCCCACUGUCCUGUGGU 78 1407ACCACAGGACAGUGGGCGG 389 1407 UUCAAAGACAACCGCACCC 79 1407UUCAAAGACAACCGCACCC 79 1425 GGGUGCGGUUGUCUUUGAA 390 1425CUGGGCGACUCCAGCGCUG 80 1425 CUGGGCGACUCCAGCGCUG 80 1443CAGCGCUGGAGUCGCCCAG 391 1443 GGCGAAAUCGCCCUGUCCA 81 1443GGCGAAAUCGCCCUGUCCA 81 1461 UGGACAGGGCGAUUUCGCC 392 1461ACGCGCAACGUGUCGGAGA 82 1461 ACGCGCAACGUGUCGGAGA 82 1479UCUCCGACACGUUGCGCGU 393 1479 ACCCGGUAUGUGUCAGAGC 83 1479ACCCGGUAUGUGUCAGAGC 83 1497 GCUCUGACACAUACCGGGU 394 1497CUGACACUGGUUCGCGUGA 84 1497 CUGACACUGGUUCGCGUGA 84 1515UCACGCGAACCAGUGUCAG 395 1515 AAGGUGGCAGAGGCUGGCC 85 1515AAGGUGGCAGAGGCUGGCC 85 1533 GGCCAGCCUCUGCCACCUU 396 1533CACUACACCAUGCGGGCCU 86 1533 CACUACACCAUGCGGGCCU 86 1551AGGCCCGCAUGGUGUAGUG 397 1551 UUCCAUGAGGAUGCUGAGG 87 1551UUCCAUGAGGAUGCUGAGG 87 1569 CCUCAGCAUCCUCAUGGAA 398 1569GUCCAGCUCUCCUUCCAGC 88 1569 GUCCAGCUCUCCUUCCAGC 88 1587GCUGGAAGGAGAGCUGGAC 399 1587 CUACAGAUCAAUGUCCCUG 89 1587CUACAGAUCAAUGUCCCUG 89 1605 CAGGGACAUUGAUCUGUAG 400 1605GUCCGAGUGCUGGAGCUAA 90 1605 GUCCGAGUGCUGGAGCUAA 90 1623UUAGCUCCAGCACUCGGAC 401 1623 AGUGAGAGCCACCCUGACA 91 1623AGUGAGAGCCACCCUGACA 91 1641 UGUCAGGGUGGCUCUCACU 402 1641AGUGGGGAACAGACAGUCC 92 1641 AGUGGGGAACAGACAGUCC 92 1659GGACUGUCUGUUCCCCACU 403 1659 CGCUGUCGUGGCCGGGGCA 93 1659CGCUGUCGUGGCCGGGGCA 93 1677 UGCCCCGGCCACGACAGCG 404 1677AUGCCCCAGCCGAACAUCA 94 1677 AUGCCCCAGCCGAACAUCA 94 1695UGAUGUUCGGCUGGGGCAU 405 1695 AUCUGGUCUGCCUGCAGAG 95 1695AUCUGGUCUGCCUGCAGAG 95 1713 CUCUGCAGGCAGACCAGAU 406 1713GACCUCAAAAGGUGUCCAC 96 1713 GACCUCAAAAGGUGUCCAC 96 1731GUGGACACCUUUUGAGGUC 407 1731 CGUGAGCUGCCGCCCACGC 97 1731CGUGAGCUGCCGCCCACGC 97 1749 GCGUGGGCGGCAGCUCACG 408 1749CUGCUGGGGAACAGUUCCG 98 1749 CUGCUGGGGAACAGUUCCG 98 1767CGGAACUGUUCCCCAGCAG 409 1767 GAAGAGGAGAGCCAGCUGG 99 1767GAAGAGGAGAGCCAGCUGG 99 1785 CCAGCUGGCUCUCCUCUUC 410 1785GAGACUAACGUGACGUACU 100 1785 GAGACUAACGUGACGUACU 100 1803AGUACGUCACGUUAGUCUC 411 1803 UGGGAGGAGGAGCAGGAGU 101 1803UGGGAGGAGGAGCAGGAGU 101 1821 ACUCCUGGUCCUCCUCCCA 412 1821UUUGAGGUGGUGAGCACAC 102 1821 UUUGAGGUGGUGAGCACAC 102 1839GUGUGCUCACCACCUCAAA 413 1839 CUGCGUCUGCAGCACGUGG 103 1839CUGCGUCUGCAGCACGUGG 103 1857 CCACGUGCUGCAGACGCAG 414 1857GAUCGGCCACUGUCGGUGC 104 1857 GAUCGGCCACUGUCGGUGC 104 1875GCACCGACAGUGGCCGAUC 415 1875 CGCUGCACGCUGCGCAACG 105 1875CGCUGCACGCUGCGCAACG 105 1893 CGUUGCGCAGCGUGCAGCG 416 1893GCUGUGGGCCAGGACACGC 106 1893 GCUGUGGGCCAGGACACGC 106 1911GCGUGUCCUGGCCCACAGC 417 1911 CAGGAGGUCAUCGUGGUGC 107 1911CAGGAGGUCAUCGUGGUGC 107 1929 GCACCACGAUGACCUCCUG 418 1929CCACACUCCUUGCCCUUUA 108 1929 CCACACUCCUUGCCCUUUA 108 1947UAAAGGGCAAGGAGUGUGG 419 1947 AAGGUGGUGGUGAUCUCAG 109 1947AAGGUGGUGGUGAUCUCAG 109 1965 CUGAGAUCACCACCACCUU 420 1965GCCAUCCUGGCCCUGGUGG 110 1965 GCCAUCCUGGCCCUGGUGG 110 1983CCACCAGGGCCAGGAUGGC 421 1983 GUGCUCACCAUCAUCUCCC 111 1983GUGCUCACCAUCAUCUCCC 111 2001 GGGAGAUGAUGGUGAGCAC 422 2001CUUAUCAUCCUCAUCAUGC 112 2001 CUUAUCAUCCUCAUCAUGC 112 2019GCAUGAUGAGGAUGAUAAG 423 2019 CUUUGGCAGAAGAAGCCAC 113 2019CUUUGGCAGAAGAAGCCAC 113 2037 GUGGCUUCUUCUGCCAAAG 424 2037CGUUACGAGAUCCGAUGGA 114 2037 CGUUACGAGAUCCGAUGGA 114 2055UCCAUCGGAUCUCGUAACG 425 2055 AAGGUGAUUGAGUCUGUGA 115 2055AAGGUGAUUGAGUCUGUGA 115 2073 UCACAGACUCAAUCACCUU 426 2073AGCUCUGACGGCCAUGAGU 116 2073 AGCUCUGACGGCCAUGAGU 116 2091ACUCAUGGCCGUCAGAGCU 427 2091 UACAUCUACGUGGACCCCA 117 2091UACAUCUACGUGGACCCCA 117 2109 UGGGGUCCACGUAGAUGUA 428 2109AUGCAGCUGCCCUAUGACU 118 2109 AUGCAGCUGCCCUAUGACU 118 2127AGUCAUAGGGCAGCUGCAU 429 2127 UCCACGUGGGAGCUGCCGC 119 2127UCCACGUGGGAGCUGCCGC 119 2145 GCGGCAGCUCCCACGUGGA 430 2145CGGGACCAGCUUGUGCUGG 120 2145 CGGGACCAGCUUGUGCUGG 120 2163CCAGCACAAGCUGGUCCCG 431 2163 GGACGCACCCUCGGCUCUG 121 2163GGACGCACCCUCGGCUCUG 121 2181 CAGAGCCGAGGGUGCGUCC 432 2181GGGGCCUUUGGGCAGGUGG 122 2181 GGGGCCUUUGGGCAGGUGG 122 2199CCACCUGCCCAAAGGCCCC 433 2199 GUGGAGGCCACGGCUCAUG 123 2199GUGGAGGCCACGGCUCAUG 123 2217 CAUGAGCCGUGGCCUCCAC 434 2217GGCCUGAGCCAUUCUCAGG 124 2217 GGCCUGAGCCAUUCUCAGG 124 2235CCUGAGAAUGGCUCAGGCC 435 2235 GCCACGAUGAAAGUGGCCG 125 2235GCCACGAUGAAAGUGGCCG 125 2253 CGGCCACUUUCAUCGUGGC 436 2253GUCAAGAUGCUUAAAUCCA 126 2253 GUCAAGAUGCUUAAAUCCA 126 2271UGGAUUUAAGCAUCUUGAC 437 2271 ACAGCCCGCAGCAGUGAGA 127 2271ACAGCCCGCAGCAGUGAGA 127 2289 UCUCACUGCUGCGGGCUGU 438 2289AAGCAAGCCCUUAUGUCGG 128 2289 AAGCAAGCCCUUAUGUCGG 128 2307CCGACAUAAGGGCUUGCUU 439 2307 GAGCUGAAGAUCAUGAGUC 129 2307GAGCUGAAGAUCAUGAGUC 129 2325 GACUCAUGAUCUUCAGCUC 440 2325CACCUUGGGCCCCACCUGA 130 2325 CACCUUGGGCCCCACCUGA 130 2343UCAGGUGGGGCCCAAGGUG 441 2343 AACGUGGUCAACCUGUUGG 131 2343AACGUGGUCAACCUGUUGG 131 2361 CCAACAGGUUGACCACGUU 442 2361GGGGCCUGCACCAAAGGAG 132 2361 GGGGCCUGCACCAAAGGAG 132 2379CUCCUUUGGUGCAGGCCCC 443 2379 GGACCCAUCUAUAUCAUCA 133 2379GGACCCAUCUAUAUCAUCA 133 2397 UGAUGAUAUAGAUGGGUCC 444 2397ACUGAGUACUGCCGCUACG 134 2397 ACUGAGUACUGCCGCUACG 134 2415CGUAGCGGCAGUACUCAGU 445 2415 GGAGACCUGGUGGACUACC 135 2415GGAGACCUGGUGGACUACC 135 2433 GGUAGUCCACCAGGUCUCC 446 2433CUGCACCGCAACAAACACA 136 2433 CUGCACCGCAACAAACACA 136 2451UGUGUUUGUUGCGGUGCAG 447 2451 ACCUUCCUGCAGCACCACU 137 2451ACCUUCCUGCAGCACCACU 137 2469 AGUGGUGCUGCAGGAAGGU 448 2469UCCGACAAGCGCCGCCCGC 138 2469 UCCGACAAGCGCCGCCCGC 138 2487GCGGGCGGCGCUUGUCGGA 449 2487 CCCAGCGCGGAGCUCUACA 139 2487CCCAGCGCGGAGCUCUACA 139 2505 UGUAGAGCUCCGCGCUGGG 450 2505AGCAAUGCUCUGCCCGUUG 140 2505 AGCAAUGCUCUGCCCGUUG 140 2523CAACGGGCAGAGCAUUGCU 451 2523 GGGCUCCCCCUGCCCAGCC 141 2523GGGCUCCCCCUGCCCAGCC 141 2541 GGCUGGGCAGGGGGAGCCC 452 2541CAUGUGUCCUUGACCGGGG 142 2541 CAUGUGUCCUUGACCGGGG 142 2559CCCCGGUCAAGGACACAUG 453 2559 GAGAGCGACGGUGGCUACA 143 2559GAGAGCGACGGUGGCUACA 143 2577 UGUAGCCACCGUCGCUCUC 454 2577AUGGACAUGAGCAAGGACG 144 2577 AUGGACAUGAGCAAGGACG 144 2595CGUCCUUGCUCAUGUCCAU 455 2595 GAGUCGGUGGACUAUGUGC 145 2595GAGUCGGUGGACUAUGUGC 145 2613 GCACAUAGUCCACCGACUC 456 2613CCCAUGCUGGACAUGAAAG 146 2613 CCCAUGCUGGACAUGAAAG 146 2631CUUUCAUGUCCAGCAUGGG 457 2631 GGAGACGUCAAAUAUGCAG 147 2631GGAGACGUCAAAUAUGCAG 147 2649 CUGCAUAUUUGACGUCUCC 458 2649GACAUCGAGUCCUCCAACU 148 2649 GACAUCGAGUCCUCCAACU 148 2667AGUUGGAGGACUCGAUGUC 459 2667 UACAUGGCCCCUUACGAUA 149 2667UACAUGGCCCCUUACGAUA 149 2685 UAUCGUAAGGGGCCAUGUA 460 2685AACUACGUUCCCUCUGCCC 150 2685 AACUACGUUCCCUCUGCCC 150 2703GGGCAGAGGGAACGUAGUU 461 2703 CCUGAGAGGACCUGCCGAG 151 2703CCUGAGAGGACCUGCCGAG 151 2721 CUCGGCAGGUCCUCUCAGG 462 2721GCAACUUUGAUCAACGAGU 152 2721 GCAACUUUGAUCAACGAGU 152 2739ACUCGUUGAUCAAAGUUGC 463 2739 UCUCCAGUGCUAAGCUACA 153 2739UCUCCAGUGCUAAGCUACA 153 2757 UGUAGCUUAGCACUGGAGA 464 2757AUGGACCUCGUGGGCUUCA 154 2757 AUGGACCUCGUGGGCUUCA 154 2775UGAAGCCCACGAGGUCCAU 465 2775 AGCUACCAGGUGGCCAAUG 155 2775AGCUACCAGGUGGCCAAUG 155 2793 CAUUGGCCACCUGGUAGCU 466 2793GGCAUGGAGUUUCUGGCCU 156 2793 GGCAUGGAGUUUCUGGCCU 156 2811AGGCCAGAAACUCCAUGCC 467 2811 UCCAAGAACUGCGUCCACA 157 2811UCCAAGAACUGCGUCCACA 157 2829 UGUGGACGCAGUUCUUGGA 468 2829AGAGACCUGGCGGCUAGGA 158 2829 AGAGACCUGGCGGCUAGGA 158 2847UCCUAGCCGCCAGGUCUCU 469 2847 AACGUGCUCAUCUGUGAAG 159 2847AACGUGCUCAUCUGUGAAG 159 2865 CUUCACAGAUGAGCACGUU 470 2865GGCAAGCUGGUCAAGAUCU 160 2865 GGCAAGCUGGUCAAGAUCU 160 2883AGAUCUUGACCAGCUUGCC 471 2883 UGUGACUUUGGCCUGGCUC 161 2883UGUGACUUUGGCCUGGCUC 161 2901 GAGCCAGGCCAAAGUCACA 472 2901CGAGACAUCAUGCGGGACU 162 2901 CGAGACAUCAUGCGGGACU 162 2919AGUCCCGGAUGAUGUCUCG 473 2919 UCGAAUUACAUCUCCAAAG 163 2919UCGAAUUACAUCUCCAAAG 163 2937 CUUUGGAGAUGUAAUUCGA 474 2937GGCAGCACCUUUUUGCCUU 164 2937 GGCAGCACCUUUUUGCCUU 164 2955AAGGCAAAAAGGUGCUGCC 475 2955 UUAAAGUGGAUGGCUCCGG 165 2955UUAAAGUGGAUGGCUCCGG 165 2973 CCGGAGCCAUCCACUUUAA 476 2973GAGAGCAUCUUCAACAGCC 166 2973 GAGAGCAUCUUCAACAGCC 166 2991GGCUGUUGAAGAUGCUCUC 477 2991 CUCUACACCACCCUGAGCG 167 2991CUCUACACCACCCUGAGCG 167 3009 CGCUCAGGGUGGUGUAGAG 478 3009GACGUGUGGUCCUUCGGGA 166 3009 GACGUGUGGUCCUUCGGGA 168 3027UCCCGAAGGACCACACGUC 479 3027 AUCCUGCUCUGGGAGAUCU 169 3027AUCCUGCUCUGGGAGAUCU 169 3045 AGAUCUCCCAGAGCAGGAU 480 3045UUCACCUUGGGUGGCACCC 170 3045 UUCACCUUGGGUGGCACCC 170 3063GGGUGCCACCCAAGGUGAA 481 3063 CCUUACCCAGAGCUGCCCA 171 3063CCUUACCCAGAGCUGCCCA 171 3081 UGGGCAGCUCUGGGUAAGG 482 3081AUGAACGAGCAGUUCUACA 172 3081 AUGAACGAGCAGUUCUACA 172 3099UGUAGAACUGCUCGUUCAU 483 3099 AAUGCCAUCAAACGGGGUU 173 3099AAUGCCAUCAAACGGGGUU 173 3117 AACCCCGUUUGAUGGCAUU 484 3117UACCGCAUGGCCCAGCCUG 174 3117 UACCGCAUGGCCCAGCCUG 174 3135CAGGCUGGGCCAUGCGGUA 485 3135 GCCCAUGCCUCCGACGAGA 175 3135GCCCAUGCCUCCGACGAGA 175 3153 UCUCGUCGGAGGCAUGGGC 486 3153AUCUAUGAGAUCAUGCAGA 176 3153 AUCUAUGAGAUCAUGCAGA 176 3171UCUGCAUGAUCUCAUAGAU 487 3171 AAGUGCUGGGAAGAGAAGU 177 3171AAGUGCUGGGAAGAGAAGU 177 3189 ACUUCUCUUCCCAGCACUU 488 3189UUUGAGAUUCGGCCCCCCU 178 3189 UUUGAGAUUCGGCCCCCCU 178 3207AGGGGGGCCGAAUCUCAAA 489 3207 UUCUCCCAGCUGGUGCUGC 179 3207UUCUCCCAGCUGGUGCUGC 179 3225 GCAGCACCAGCUGGGAGAA 490 3225CUUCUCGAGAGACUGUUGG 180 3225 CUUCUCGAGAGACUGUUGG 180 3243CCAACAGUCUCUCGAGAAG 491 3243 GGCGAAGGUUACAAAAAGA 181 3243GGCGAAGGUUACAAAAAGA 181 3261 UCUUUUUGUAACCUUCGCC 492 3261AAGUACCAGCAGGUGGAUG 182 3261 AAGUACCAGCAGGUGGAUG 182 3279CAUCCACCUGCUGGUACUU 493 3279 GAGGAGUUUCUGAGGAGUG 183 3279GAGGAGUUUCUGAGGAGUG 183 3297 CACUCCUCAGAAACUCCUC 494 3297GACCACCCAGCCAUCCUUC 184 3297 GACCACCCAGCCAUCCUUC 184 3315GAAGGAUGGCUGGGUGGUC 495 3315 CGGUCCCAGGCCCGCUUGC 185 3315CGGUCCCAGGCCCGCUUGC 185 3333 GCAAGCGGGCCUGGGACCG 496 3333CCUGGGUUCCAUGGCCUCC 186 3333 CCUGGGUUCCAUGGCCUCC 186 3351GGAGGCCAUGGAACCCAGG 497 3351 CGAUCUCCCCUGGACACCA 187 3351CGAUCUCCCCUGGACACCA 187 3369 UGGUGUCCAGGGGAGAUCG 498 3369AGCUCCGUCCUCUAUACUG 188 3369 AGCUCCGUCCUCUAUACUG 188 3387CAGUAUAGAGGACGGAGCU 499 3387 GCCGUGCAGCCCAAUGAGG 189 3387GCCGUGCAGCCCAAUGAGG 189 3405 CCUCAUUGGGCUGCACGGC 500 3405GGUGACAACGACUAUAUCA 190 3405 GGUGACAACGACUAUAUCA 190 3423UGAUAUAGUCGUUGUCACC 501 3423 AUCCCCCUGCCUGACCCCA 191 3423AUCCCCCUGCCUGACCCCA 191 3441 UGGGGUCAGGCAGGGGGAU 502 3441AAACCCGAGGUUGCUGACG 192 3441 AAACCCGAGGUUGCUGACG 192 3459CGUCAGCAACCUCGGGUUU 503 3459 GAGGGCCCACUGGAGGGUU 193 3459GAGGGCCCACUGGAGGGUU 193 3477 AACCCUCCAGUGGGCCCUC 504 3477UCCCCCAGCCUAGCCAGCU 194 3477 UCCCCCAGCCUAGCCAGCU 194 3495AGCUGGCUAGGCUGGGGGA 505 3495 UCCACCCUGAAUGAAGUCA 195 3495UCCACCCUGAAUGAAGUCA 195 3513 UGACUUCAUUCAGGGUGGA 506 3513AACACCUCCUCAACCAUCU 196 3513 AACACCUCCUCAACCAUCU 196 3531AGAUGGUUGAGGAGGUGUU 507 3531 UCCUGUGACAGCCCCCUGG 197 3531UCCUGUGACAGCCCCCUGG 197 3549 CCAGGGGGCUGUCACAGGA 508 3549GAGCCCCAGGACGAACCAG 198 3549 GAGCCCCAGGACGAACCAG 198 3567CUGGUUCGUCCUGGGGCUC 509 3567 GAGCCAGAGCCCCAGCUUG 199 3567GAGCCAGAGCCCCAGCUUG 199 3585 CAAGCUGGGGCUCUGGCUC 510 3585GAGCUCCAGGUGGAGCCGG 200 3585 GAGCUCCAGGUGGAGCCGG 200 3663CCGGCUCCACCUGGAGCUC 511 3603 GAGCCAGAGCUGGAACAGU 201 3603GAGCCAGAGCUGGAACAGU 201 3621 ACUGUUCCAGCUCUGGCUC 512 3621UUGCCGGAUUCGGGGUGCC 202 3621 UUGCCGGAUUCGGGGUGCC 202 3639GGCACCCCGAAUCCGGCAA 513 3639 CCUGCGCCUCGGGCGGAAG 203 3639CCUGCGCCUCGGGCGGAAG 203 3657 CUUCCGCCCGAGGCGCAGG 514 3657GCAGAGGAUAGCUUCCUGU 204 3657 GCAGAGGAUAGCUUCCUGU 204 3675ACAGGAAGCUAUCCUCUGC 515 3675 UAGGGGGCUGGCCCCUACC 205 3675UAGGGGGCUGGCCCCUACC 205 3693 GGUAGGGGCCAGCCCCCUA 516 3693CCUGCCCUGCCUGAAGCUC 206 3693 CCUGCCCUGCCUGAAGCUC 206 3711GAGCUUCAGGCAGGGCAGG 517 3711 CCCCCCCUGCCAGCACCCA 207 3711CCCCCCCUGCCAGCACCCA 207 3729 UGGGUGCUGGCAGGGGGGG 518 3729AGCAUCUCCUGGCCUGGCC 208 3729 AGCAUCUCCUGGCCUGGCC 208 3747GGCCAGGCCAGGAGAUGCU 519 3747 CUGACCGGGCUUCCUGUCA 209 3747CUGACCGGGCUUCCUGUCA 209 3765 UGACAGGAAGCCCGGUCAG 520 3765AGCCAGGCUGCCCUUAUCA 210 3765 AGCCAGGCUGCCCUUAUCA 210 3783UGAUAAGGGCAGCCUGGCU 521 3783 AGCUGUCCCCUUCUGGAAG 211 3783AGCUGUCCCCUUCUGGAAG 211 3801 CUUCCAGAAGGGGACAGCU 522 3801GCUUUCUGCUCCUGACGUG 212 3801 GCUUUCUGCUCCUGACGUG 212 3819CACGUCAGGAGCAGAAAGC 523 3819 GUUGUGCCCCAAACCCUGG 213 3819GUUGUGCCCCAAACCCUGG 213 3837 CCAGGGUUUGGGGCACAAC 524 3837GGGCUGGCUUAGGAGGCAA 214 3837 GGGCUGGCUUAGGAGGCAA 214 3855UUGCCUCCUAAGCCAGCCC 525 3855 AGAAAACUGCAGGGGCCGU 215 3855AGAAAACUGCAGGGGCCGU 215 3873 ACGGCCCCUGCAGUUUUCU 526 3873UGACCAGCCCUCUGCCUCC 216 3873 UGACCAGCCCUCUGCCUCC 216 3891GGAGGCAGAGGGCUGGUCA 527 3891 CAGGGAGGCCAACUGACUC 217 3891CAGGGAGGCCAACUGACUC 217 3909 GAGUCAGUUGGCCUCCCUG 528 3909CUGAGCCAGGGUUCCCCCA 218 3909 CUGAGCCAGGGUUCCCCCA 218 3927UGGGGGAACCCUGGCUCAG 529 3927 AGGGAACUCAGUUUUCCCA 219 3927AGGGAACUCAGUUUUCCCA 219 3945 UGGGAAAACUGAGUUCCCU 530 3945AUAUGUAAGAUGGGAAAGU 220 3945 AUAUGUAAGAUGGGAAAGU 220 3963ACUUUCCCAUCUUACAUAU 531 3963 UUAGGCUUGAUGACCCAGA 221 3963UUAGGCUUGAUGACCCAGA 221 3981 UCUGGGUCAUCAAGCCUAA 532 3981AAUCUAGGAUUCUCUCCCU 222 3981 AAUCUAGGAUUCUCUCCCU 222 3999AGGGAGAGAAUCCUAGAUU 533 3999 UGGCUGACAGGUGGGGAGA 223 3999UGGCUGACAGGUGGGGAGA 223 4017 UCUCCCCACCUGUCAGCCA 534 4017ACCGAAUCCCUCCCUGGGA 224 4017 ACCGAAUCCCUCCCUGGGA 224 4035UCCCAGGGAGGGAUUCGGU 535 4035 AAGAUUCUUGGAGUUACUG 225 4035AAGAUUCUUGGAGUUACUG 225 4053 CAGUAACUCCAAGAAUCUU 536 4053GAGGUGGUAAAUUAACUUU 226 4053 GAGGUGGUAAAUUAACUUU 226 4071AAAGUUAAUUUACCACCUC 537 4071 UUUUCUGUUCAGCCAGCUA 227 4071UUUUCUGUUCAGCCAGCUA 227 4089 UAGCUGGCUGAACAGAAAA 538 4089ACCCCUCAAGGAAUCAUAG 228 4089 ACCCCUCAAGGAAUCAUAG 228 4107CUAUGAUUCCUUGAGGGGU 539 4107 GCUCUCUCCUCGCACUUUU 229 4107GCUCUCUCCUCGCACUUUU 229 4125 AAAAGUGCGAGGAGAGAGC 540 4125UUAUCCACCCAGGAGCUAG 230 4125 UUAUCCACCCAGGAGCUAG 230 4143CUAGCUCCUGGGUGGAUAA 541 4143 GGGAAGAGACCCUAGCCUC 231 4143GGGAAGAGACCCUAGCCUC 231 4161 GAGGCUAGGGUCUCUUCCC 542 4161CCCUGGCUGCUGGCUGAGC 232 4161 CCCUGGCUGCUGGCUGAGC 232 4179GCUCAGCCAGCAGCCAGGG 543 4179 CUAGGGCCUAGCCUUGAGC 233 4179CUAGGGCCUAGCCUUGAGC 233 4197 GCUCAAGGCUAGGCCCUAG 544 4197CAGUGUUGCCUCAUCCAGA 234 4197 CAGUGUUGCCUCAUCCAGA 234 4215UCUGGAUGAGGCAACACUG 545 4215 AAGAAAGCCAGUCUCCUCC 235 4215AAGAAAGCCAGUCUCCUCC 235 4233 GGAGGAGACUGGCUUUCUU 546 4233CCUAUGAUGCCAGUCCCUG 236 4233 CCUAUGAUGCCAGUCCCUG 236 4251CAGGGACUGGCAUCAUAGG 547 4251 GCGUUCCCUGGCCCGAGCU 237 4251GCGUUCCCUGGCCCGAGCU 237 4269 AGCUCGGGCCAGGGAACGC 548 4269UGGUCUGGGGCCAUUAGGC 238 4269 UGGUCUGGGGCCAUUAGGC 238 4287GCCUAAUGGCCCCAGACCA 549 4287 CAGCCUAAUUAAUGCUGGA 239 4287CAGCCUAAUUAAUGCUGGA 239 4305 UCCAGCAUUAAUUAGGCUG 550 4305AGGCUGAGCCAAGUACAGG 240 4305 AGGCUGAGCCAAGUACAGG 240 4323CCUGUACUUGGCUCAGCCU 551 4323 GACACCCCCAGCCUGCAGC 241 4323GACACCCCCAGCCUGCAGC 241 4341 GCUGCAGGCUGGGGGUGUC 552 4341CCCUUGCCCAGGGCACUUG 242 4341 CCCUUGCCCAGGGCACUUG 242 4359CAAGUGCCCUGGGCAAGGG 553 4359 GGAGCACACGCAGCCAUAG 243 4359GGAGCACACGCAGCCAUAG 243 4377 CUAUGGCUGCGUGUGCUCC 554 4377GCAAGUGCCUGUGUCCCUG 244 4377 GCAAGUGCCUGUGUCCCUG 244 4395CAGGGACACAGGCACUUGC 555 4395 GUCCUUCAGGCCCAUCAGU 245 4395GUCCUUCAGGCCCAUCAGU 245 4413 ACUGAUGGGCCUGAAGGAC 556 4413UCCUGGGGCUUUUUCUUUA 246 4413 UCCUGGGGCUUUUUCUUUA 246 4431UAAAGAAAAAGCCCCAGGA 557 4431 AUCACCCUCAGUCUUAAUC 247 4431AUCACCCUCAGUCUUAAUC 247 4449 GAUUAAGACUGAGGGUGAU 558 4449CCAUCCACCAGAGUCUAGA 248 4449 CCAUCCACCAGAGUCUAGA 248 4467UCUAGACUCUGGUGGAUGG 559 4467 AAGGCCAGACGGGCCCCGC 249 4467AAGGCCAGACGGGCCCCGC 249 4485 GCGGGGCCCGUCUGGCCUU 560 4485CAUCUGUGAUGAGAAUGUA 250 4485 CAUCUGUGAUGAGAAUGUA 250 4503UACAUUCUCAUCACAGAUG 561 4503 AAAUGUGCCAGUGUGGAGU 251 4503AAAUGUGCCAGUGUGGAGU 251 4521 ACUCCACACUGGCACAUUU 562 4521UGGCCACGUGUGUGUGCCA 252 4521 UGGCCACGUGUGUGUGCCA 252 4539UGGCACACACACGUGGCCA 563 4539 AGUAUAUGGCCCUGGCUCU 253 4539AGUAUAUGGCCCUGGCUCU 253 4557 AGAGCCAGGGCCAUAUACU 564 4557UGCAUUGGACCUGCUAUGA 254 4557 UGCAUUGGACCUGCUAUGA 254 4575UCAUAGCAGGUCCAAUGCA 565 4575 AGGCUUUGGAGGAAUCCCU 255 4575AGGCUUUGGAGGAAUCCCU 255 4593 AGGGAUUCCUCCAAAGCCU 566 4593UCACCCUCUCUGGGCCUCA 256 4593 UCACCCUCUCUGGGCCUCA 256 4611UGAGGCCCAGAGAGGGUGA 567 4611 AGUUUCCCCUUCAAAAAAU 257 4611AGUUUCCCCUUCAAAAAAU 257 4629 AUUUUUUGAAGGGGAAACU 568 4629UGAAUAAGUCGGACUUAUU 258 4629 UGAAUAAGUCGGACUUAUU 258 4647AAUAAGUCCGACUUAUUCA 569 4647 UAACUCUGAGUGCCUUGCC 259 4647UAACUCUGAGUGCCUUGCC 259 4665 GGCAAGGCACUCAGAGUUA 570 4665CAGCACUAACAUUCUAGAG 260 4665 CAGCACUAACAUUCUAGAG 260 4683CUCUAGAAUGUUAGUGCUG 571 4683 GUAUUCCAGGUGGUUGCAC 261 4683GUAUUCCAGGUGGUUGCAC 261 4701 GUGCAACCACCUGGAAUAC 572 4701CAUUUGUCCAGAUGAAGCA 262 4701 CAUUUGUCCAGAUGAAGCA 262 4719UGCUUCAUCUGGACAAAUG 573 4719 AAGGCCAUAUACCCUAAAC 263 4719AAGGCCAUAUACCCUAAAC 263 4737 GUUUAGGGUAUAUGGCCUU 574 4737CUUCCAUCCUGGGGGUCAG 264 4737 CUUCCAUCCUGGGGGUCAG 264 4755CUGACCCCCAGGAUGGAAG 575 4755 GCUGGGCUCCUGGGAGAUU 265 4755GCUGGGCUCCUGGGAGAUU 265 4773 AAUCUCCCAGGAGCCCAGC 576 4773UCCAGAUCACACAUCACAC 266 4773 UCCAGAUCACACAUCACAC 266 4791GUGUGAUGUGUGAUCUGGA 577 4791 CUCUGGGGACUCAGGAACC 267 4791CUCUGGGGACUCAGGAACC 267 4809 GGUUCCUGAGUCCCCAGAG 578 4809CAUGCCCCUUCCCCAGGCC 268 4809 CAUGCCCCUUCCCCAGGCC 268 4827GGCCUGGGGAAGGGGCAUG 579 4827 CCCCAGCAAGUCUCAAGAA 269 4827CCCCAGCAAGUCUCAAGAA 269 4845 UUCUUGAGACUUGCUGGGG 580 4845ACACAGCUGCACAGGCCUU 270 4845 ACACAGCUGCACAGGCCUU 270 4863AAGGCCUGUGCAGCUGUGU 581 4863 UGACUUAGAGUGACAGCCG 271 4863UGACUUAGAGUGACAGCCG 271 4881 CGGCUGUCACUCUAAGUCA 582 4881GGUGUCCUGGAAAGCCCCA 272 4881 GGUGUCCUGGAAAGCCCCA 272 4899UGGGGCUUUCCAGGACACC 583 4899 AAGCAGCUGCCCCAGGGAC 273 4899AAGCAGCUGCCCCAGGGAC 273 4917 GUCCCUGGGGCAGCUGCUU 584 4917CAUGGGAAGACCACGGGAC 274 4917 CAUGGGAAGACCACGGGAC 274 4935GUCCCGUGGUCUUCCCAUG 585 4935 CCUCUUUCACUACCCACGA 275 4935CCUCUUUCACUACCCACGA 275 4953 UCGUGGGUAGUGAAAGAGG 586 4953AUGACCUCCGGGGGUAUCC 276 4953 AUGACCUCCGGGGGUAUCC 276 4971GGAUACCCCCGGAGGUCAU 587 4971 CUGGGCAAAAGGGACAAAG 277 4971CUGGGCAAAAGGGACAAAG 277 4989 CUUUGUCCCUUUUGCCCAG 588 4989GAGGGCAAAUGAGAUCACC 278 4989 GAGGGCAAAUGAGAUCACC 278 5007GGUGAUCUCAUUUGCCCUC 589 5007 CUCCUGCAGCCCACCACUC 279 5007CUCCUGCAGCCCACCACUC 279 5025 GAGUGGUGGGCUGCAGGAG 590 5025CCAGCACCUGUGCCGAGGU 280 5025 CCAGCACCUGUGCCGAGGU 280 5043ACCUCGGCACAGGUGCUGG 591 5043 UCUGCGUCGAAGACAGAAU 281 5043UCUGCGUCGAAGACAGAAU 281 5061 AUUCUGUCUUCGACGCAGA 592 5061UGGACAGUGAGGACAGUUA 282 5061 UGGACAGUGAGGACAGUUA 282 5079UAACUGUCCUCACUGUCCA 593 5079 AUGUCUUGUAAAAGACAAG 283 5079AUGUCUUGUAAAAGACAAG 283 5097 CUUGUCUUUUACAAGACAU 594 5097GAAGCUUCAGAUGGUACCC 284 5097 GAAGCUUCAGAUGGUACCC 284 5115GGGUACCAUCUGAAGCUUC 595 5115 CCAAGAAGGAUGUGAGAGG 285 5115CCAAGAAGGAUGUGAGAGG 285 5133 CCUCUCACAUCCUUCUUGG 596 5133GUGGCCGCUUGGAGUUUGC 286 5133 GUGGCCGCUUGGAGUUUGC 286 5151GCAAACUCCAAGCGGCCAC 597 5151 CCCCUCACCCACCAGCUGC 287 5151CCCCUCACCCACCAGCUGC 287 5169 GCAGCUGGUGGGUGAGGGG 598 5169CCCCAUCCCUGAGGCAGCG 288 5169 CCCCAUCCCUGAGGCAGCG 288 5187CGCUGCCUCAGGGAUGGGG 599 5187 GCUCCAUGGGGGUAUGGUU 289 5187GCUCCAUGGGGGUAUGGUU 289 5205 AACCAUACCCCCAUGGAGC 600 5205UUUGUCACUGCCCAGACCU 290 5205 UUUGUCACUGCCCAGACCU 290 5223AGGUCUGGGCAGUGACAAA 601 5223 UAGCAGUGACAUCUCAUUG 291 5223UAGCAGUGACAUCUCAUUG 291 5241 CAAUGAGAUGUCACUGCUA 602 5241GUCCCCAGCCCAGUGGGCA 292 5241 GUCCCCAGCCCAGUGGGCA 292 5259UGCCCACUGGGCUGGGGAC 603 5259 AUUGGAGGUGCCAGGGGAG 293 5259AUUGGAGGUGCCAGGGGAG 293 5277 CUCCCCUGGCACCUCCAAU 604 5277GUCAGGGUUGUAGCCAAGA 294 5277 GUCAGGGUUGUAGCCAAGA 294 5295UCUUGGCUACAACCCUGAC 605 5295 ACGCCCCCGCACGGGGAGG 295 5295ACGCCCCCGCACGGGGAGG 295 5313 CCUCCCCGUGCGGGGGCGU 606 5313GGUUGGGAAGGGGGUGCAG 296 5313 GGUUGGGAAGGGGGUGCAG 296 5331CUGCACCCCCUUCCCAACC 607 5331 GGAAGCUCAACCCCUCUGG 297 5331GGAAGCUCAACCCCUCUGG 297 5349 CCAGAGGGGUUGAGCUUCC 608 5349GGCACCAACCCUGCAUUGC 298 5349 GGCACCAACCCUGCAUUGC 298 5367GCAAUGCAGGGUUGGUGCC 609 5367 CAGGUUGGCACCUUACUUC 299 5367CAGGUUGGCACCUUACUUC 299 5385 GAAGUAAGGUGCCAACCUG 610 5385CCCUGGGAUCCCCAGAGUU 300 5385 CCCUGGGAUCCCCAGAGUU 300 5403AACUCUGGGGAUCCCAGGG 611 5403 UGGUCCAAGGAGGGAGAGU 301 5403UGGUCCAAGGAGGGAGAGU 301 5421 ACUCUCCCUCCUUGGACCA 612 5421UGGGUUCUCAAUACGGUAC 302 5421 UGGGUUCUCAAUACGGUAC 302 5439GUACCGUAUUGAGAACCCA 613 5439 CCAAAGAUAUAAUCACCUA 303 5439CCAAAGAUAUAAUCACCUA 303 5457 UAGGUGAUUAUAUCUUUGG 614 5457AGGUUUACAAAUAUUUUUA 304 5457 AGGUUUACAAAUAUUUUUA 304 5475UAAAAAUAUUUGUAAACCU 615 5475 AGGACUCACGUUAACUCAC 305 5475AGGACUCACGUUAACUCAC 305 5493 GUGAGUUAACGUGAGUCCU 616 5493CAUUUAUACAGCAGAAAUG 306 5493 CAUUUAUACAGCAGAAAUG 306 5511CAUUUCUGCUGUAUAAAUG 617 5511 GCUAUUUUGUAUGCUGUUA 307 5511GCUAUUUUGUAUGCUGUUA 307 5529 UAACAGCAUACAAAAUAGC 618 5529AAGUUUUUCUAUCUGUGUA 308 5529 AAGUUUUUCUAUCUGUGUA 308 5547UACACAGAUAGAAAAACUU 619 5547 ACUUUUUUUUAAGGGAAAG 309 5547ACUUUUUUUUAAGGGAAAG 309 5565 CUUUCCCUUAAAAAAAAGU 620 5565GAUUUUAAUAUUAAACCUG 310 5565 GAUUUUAAUAUUAAACCUG 310 5583CAGGUUUAAUAUUAAAAUC 621 5578 AACCUGGUGCUUCUCACUC 311 5578AACCUGGUGCUUCUCACUC 311 5596 GAGUGAGAAGCACCAGGUU 622 The 3′-ends of theUpper sequence and the Lower sequence of the siNA construct can includean overhang sequence, for example about 1, 2, 3, or 4 nucleotides inlength, preferably 2 nucleotides in length, wherein the overhangingsequence of the lower sequence is optionally complementary to a portionof the target sequence. The upper sequence is also referred to as thesense strand, whereas the lower sequence is also referred to as theantisense strand. The upper and lower sequences in the Table can furthercomprise a chemical modification having Formulae I-VII, such asexemplary siNA constructs shown in FIGS. 4 and 5, or havingmodifications described in Table IV or any combination thereof.

TABLE III PDGFRB Synthetic Modified siNA Constructs Target Seq Seq PosTarget ID Cmpd# Aliases Sequence ID 422 UGCCUGUCCUUCUACUCAGCUGU 62331910 PDGFRB:208U21 sense siNA CCUGUCCUUCUACUCAGCUTT 631 427GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:949U21 sense siNAAGGUGGAUUCUGAUGCCUATT 632 506 GGCACACUACAAUUUGCUGAGCU 625 PDGFRB:1325U21sense siNA CACACUACAAUUUGCUGAGTT 633 511 CGAGUGCUGGAGCUAAGUGAGAG 626PDGFRB:1610U21 sense siNA AGUGCUGGAGCUAAGUGAGTT 634 616CUCGAAUUACAUCUCCAAAGGCA 627 31911 PDGFRB:2920U21 sense siNACGAAUUACAUCUCCAAAGGTT 635 681 CUGCUAUGAGGCUUUGGAGGAAU 628 31912PDGFRB:4569U21 sense siNA GCUAUGAGGCUUUGGAGGATT 636 751GACAAAGAGGGCAAAUGAGAUCA 629 31913 PDGFRB:4985U21 sense siNACAAAGAGGGCAAAUGAGAUTT 637 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5415U21sense siNA GGAGAGUGGGUUCUCAAUATT 638 422 UGCCUGUCCUUCUACUCAGCUGU 62331914 PDGFRB:226L21 antisense siNA AGCUGAGUAGAAGGACAGGTT 639 (208C) 427GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAUAGGCAUCAGAAUCCACCUTT 640 (949C) 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1343L21 antisense siNA CUCAGCAAAUUGUAGUGUGTT 641 (1325C) 511CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNACUCACUUAGCUCCAGCACUTT 642 (1610C) 616 CUCGAAUUAGAUCUCCAAAGGCA 627 31915PDGFRB:2938L21 antisense siNA CCUUUGGAGAUGUAAUUCGTT 643 (2920C) 681CUGCUAUGAGGCUUUGGAGGAAU 628 31916 PDGFRB:4587L21 antisense siNAUCCUCCAAAGCCUCAUAGCTT 644 (4569C) 751 GACAAAGAGGGCAAAUGAGAUCA 629 31917PDGFRB:5003L21 antisense siNA AUCUCAUUUGCCCUCUUUGTT 645 (4985C) 815AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAUAUUGAGAACCCACUCUCCTT 646 (5415C) 422 UGCCUGUCCUUCUACUCAGCUGU 623PDGFRB:208U21 sense siNA stab04 B ccuGuccuucuAcucAGcuTT B 647 427GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:949U21 sense siNA stab04 BAGGuGGAuucuGAuGccuATT B 648 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1325U21 sense siNA stab04 B cAcAcuAcAAuuuGcuGAGTT B 649 511CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1610U21 sense siNA stab04 BAGuGcuGGAGcuAAGuGAGTT B 650 616 CUCGAAUUACAUCUCCAAAGGCA 627PDGFRB:2920U21 sense siNA stab04 B cGAAuuAcAucuccAAAGGTT B 651 681CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4569U21 sense siNA stab04 BGcuAuGAGGcuuuGGAGGATT B 652 751 GACAAAGAGGGCAAAUGAGAUCA 629PDGFRB:4985U21 sense siNA stab04 B cAAAGAGGGcAAAuGAGAuTT B 653 815AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5415U21 sense siNA stab04 BGGAGAGuGGGuucucAAuATT B 654 422 UGCCUGUCCUUCUACUCAGCUGU 623PDGFRB:226L21 antisense siNA AGcuGAGuAGAAGGAcAGGTsT 655 (208C) stab05427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAuAGGcAucAGAAuccAccuTsT 656 (949C) stab05 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1343L21 antisense siNA cucAGcAAAuuGuAGuGuGTsT 657 (1325C) stab05511 CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNAcucAcuuAGcuccAGcAcuTsT 658 (1610C) stab05 616 CUCGAAUUACAUCUCCAAAGGCA627 PDGFRB:2938L21 antisense siNA ccuuuGGAGAuGuAAuucGTsT 659 (2920C)stab05 681 CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNAuccuccAAAGccucAuAGcTsT 660 (4569C) stab05 751 GACAAAGAGGGCAAAUGAGAUCA629 PDGFRB:5003L21 antisense siNA AucucAuuuGcccucuuuGTsT 661 (4985C)stab05 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAuAuuGAGAAcccAcucuccTsT 662 (5415C) stab05 422 UGCCUGUCCUUCUACUCAGCUGU623 PDGFRB:208U21 sense siNA stab07 B ccuGuccuucuAcucAGcuTT B 663 427GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:949U21 sense siNA stab07 BAGGuGGAuucuGAuGccuATT B 664 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1325U21 sense siNA stab07 B cAcAcuAcAAuuuGcuGAGTT B 665 511CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1610U21 sense siNA stab07 BAGuGcuGGAGcuAAGuGAGTT B 666 616 CUCGAAUUACAUCUCCAAAGGCA 627PDGFRB:2920U21 sense siNA stab07 B cGAAuuAcAucuccAAAGGTT B 667 681CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4569U21 sense siNA stab07 BGcuAuGAGGcuuuGGAGGATT B 668 751 GACAAAGAGGGCAAAUGAGAUCA 629PDGFRB:4985U21 sense siNA stab07 B cAAAGAGGGcAAAuGAGAuTT B 669 815AGGGAGAGUGGGUUGUCAAUACG 630 PDGFRB:5415U21 sense siNA stab07 BGGAGAGuGGGuucucAAuATT B 670 422 UGCCUGUCCUUCUACUCAGCUGU 623PDGFRB:226L21 antisense siNA AGcuGAGuAGAAGGAcAGGTsT 671 (208C) stab11427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAuAGGcAucAGAAuccAccuTsT 672 (949C) stab11 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1343L21 antisense siNA cucAGcAAAuuGuAGuGuGTsT 673 (1325C) stab11511 CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNAcucAcuuAGcuccAGcAcuTsT 674 (1610C) stab11 616 CUCGAAUUACAUCUCCAAAGGCA627 PDGFRB:2938L21 antisense siNA ccuuuGGAGAuGuAAuucGTsT 675 (2920C)stab11 681 CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNAuccuccAAAGccucAuAGcTsT 676 (4569C) stab11 751 GACAAAGAGGGCAAAUGAGAUCA629 PDGFRB:5003L21 antisense siNA AucucAuuuGcccucuuuGTsT 677 (4985C)stab11 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAuAuuGAGAAcccAcucuccTsT 678 (5415C) stab11 422 UGCCUGUCCUUCUACUCAGCUGU623 PDGFRB:208U21 sense siNA stab18 B ccuGuccuucuAcucAGcuTT B 679 427GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:949U21 sense siNA stab18 BAGGuGGAuucuGAuGccuATT B 680 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1325U21 sense siNA stab18 B cAcAcuAcAAuuuGcuGAGTT B 681 511CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1610U21 sense siNA stab18 BAGuGcuGGAGcuAAGuGAGTT B 682 616 CUCGAAUUACAUCUCCAAAGGCA 627PDGFRB:2920U21 sense siNA stab18 B cGAAuuAcAucuccAAAGGTT B 683 681CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4569U21 sense siNA stab18 BGcuAuGAGGcuuuGGAGGATT B 684 751 GACAAAGAGGGCAAAUGAGAUCA 629PDGFRB:4985U21 sense siNA stab18 B cAAAGAGGGcAAAuGAGAuTT B 685 815AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5415U21 sense siNA stab18 BGGAGAGuGGGuucucAAuATT B 686 422 UGCCUGUCCUUCUACUCAGCUGU 623PDGFRB:226L21 antisense siNA AGcuGAGuAGAAGGAcAGGTsT 687 (208C) stab08427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAuAGGcAucAGAAuccAccuTsT 688 (949C) stab08 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1343L21 antisense siNA cucAGcAAAuuGuAGuGuGTsT 689 (1325C) stab08511 CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNAcucAcuuAGcuccAGcAcuTsT 690 (1610C) stab08 616 CUCGAAUUACAUCUCCAAAGGCA627 PDGFRB:2938L21 antisense siNA ccuuuGGAGAuGuAAuucGTsT 691 (2920C)stab08 681 CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNAuccuccAAAGccucAuAGcTsT 692 (4569C) stab08 751 GACAAAGAGGGCAAAUGAGAUCA629 PDGFRB:5003L21 antisense siNA AucucAuuuGcccucuuuGTsT 693 (4985C)stab08 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAuAuuGAGAAcccAcucuccTsT 694 (5415C) stab08 422 UGCCUGUCCUUCUACUCAGCUGU623 37092 PDGFRB:208U21 sense siNA stab09 B CCUGUCCUUCUACUCAGCUTT B 695427 GGAGGUGGAUUCUGAUGCCUACU 624 37093 PDGFRB:949U21 sense siNA stab09 BAGGUGGAUUCUGAUGCCUATT B 696 506 GGCACACUACAAUUUGCUGAGCU 625 37094PDGFRB:1325U21 sense siNA stab09 B CACACUACAAUUUGCUGAGTT B 697 511CGAGUGCUGGAGCUAAGUGAGAG 626 37095 PDGFRB:1610U21 sense siNA stab09 BAGUGCUGGAGCUAAGUGAGTT B 698 616 CUCGAAUUACAUCUCCAAAGGCA 627 37096PDGFRB:2920U21 sense siNA stab09 B CGAAUUACAUCUCCAAAGGTT B 699 681CUGCUAUGAGGCUUUGGAGGAAU 628 37097 PDGFRB:4569U21 sense siNA stab09 BGCUAUGAGGCUUUGGAGGATT B 700 751 GACAAAGAGGGCAAAUGAGAUCA 629 37098PDGFRB:4985U21 sense siNA stab09 B CAAAGAGGGCAAAUGAGAUTT B 701 815AGGGAGAGUGGGUUCUCAAUACG 630 37099 PDGFRB:5415U21 sense siNA stab09 BGGAGAGUGGGUUCUCAAUATT B 702 422 UGCCUGUCCUUCUACUCAGCUGU 623PDGFRB:226L21 antisense siNA AGCUGAGUAGAAGGACAGGTsT 703 (208C) stab10427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAUAGGCAUCAGAAUCCACCUTsT 704 (949C) stab10 506 GGCACACUACAAUUUGCUGAGCU 625PDGFRB:1343L21 antisense siNA CUCAGCAAAUUGUAGUGUGTsT 705 (1325C) stab10511 CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNACUCACUUAGCUCCAGCACUTsT 706 (1610C) stab10 616 CUCGAAUUACAUCUC0AAAGGCA627 PDGFRB:2938L21 antisense siNA CCUUUGGAGAUGUAAUUCGTsT 707 (2920C)stab10 681 CUGCUAUGAGGCUUUGGAGGAAU 626 PDGFRB:4587L21 antisense siNAUCCUCCAAAGCCUCAUAGCTsT 708 (4569C) stab10 751 GACAAAGAGGGCAAAUGAGAUCA629 PDGFRB:5003L21 antisense siNA AUCUCAUUUGCCCUCUUUGTsT 709 (4985C)stab10 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAUAUUGAGAACCCACUCUCCTsT 710 (5415C) stab10 422 UGCCUGUCCUUCUACUCAGCUGU623 PDGFRB:226L21 antisense siNA AGcuGAGuAGAAGGAcAGGTT B 711 (208C)stab19 427 GGAGGUGGAUUCUGAUGCCUACU 624 PDGFRB:967L21 antisense siNAuAGGcAucAGAAuCcAccuTT B 712 (949C) stab19 506 GGCACACUACAAUUUGCUGAGCU625 PDGFRB:1343L21 antisense siNA cucAGcAAAuuGuAGuGuGTT B 713 (1325C)stab19 511 CGAGUGCUGGAGCUAAGUGAGAG 626 PDGFRB:1628L21 antisense siNAcucAcuuAGcuccAGcAcuTT B 714 (1610C) stab19 616 CUCGAAUUACAUCUCCAAAGGCA627 PDGFRB:2938L21 antisense siNA ccuuuGGAGAuGuAAuucGTT B 715 (2920C)stab19 681 CUGCUAUGAGGCUUUGGAGGAAU 628 PDGFRB:4587L21 antisense siNAuccuccAAAGccucAuAGcTT B 716 (4569C) stab19 751 GACAAAGAGGGCAAAUGAGAUCA629 PDGFRB:5003L21 antisense siNA AucucAuuuGcccucuuuGTT B 717 (4985C)stab19 815 AGGGAGAGUGGGUUCUCAAUACG 630 PDGFRB:5433L21 antisense siNAuAuuGAGAAcccAcucuccTT B 718 (5415C) stab19 422 UGCCUGUCCUUCUACUCAGCUGU623 37100 PDGFRB:226L21 antisense siNA AGCUGAGUAGAAGGACAGGTT B 719(208C) stab22 427 GGAGGUGGAUUCUGAUGCCUACU 624 37101 PDGFRB:967L21antisense siNA UAGGCAUCAGAAUCCACCUTT B 720 (949C) stab22 506GGCACACUACAAUUUGCUGAGCU 625 37102 PDGFRB:1343121 antisense siNACUCAGCAAAUUGUAGUGUGTT B 721 (1325C) stab22 511 CGAGUGCUGGAGCUAAGUGAGAG626 37103 PDGFRB:1628L21 antisense siNA CUCACUUAGCUCCAGCACUTT B 722(1610C) stab22 616 CUCGAAUUACAUCUCCAAAGGCA 627 37104 PDGFRB:2938L21antisense siNA CCUUUGGAGAUGUAAUUCGTT B 723 (2920C) stab22 681CUGCUAUGAGGCUUUGGAGGAAU 628 37105 PDGFRB:4587L21 antisense siNAUCCUCCAAAGCCUCAUAGCTT B 724 (4569C) stab22 751 GACAAAGAGGGCAAAUGAGAUCA629 37106 PDGFRB:5003L21 antisense siNA AUCUCAUUUGCCCUCUUUGTT B 725(4985C) stab22 815 AGGGAGAGUGGGUUCUCAAUACG 630 37107 PDGFRB:5433L21antisense siNA UAUUGAGAACCCACUCUCCTT B 726 (5415C) stab22 Uppercase= ribonucleotide u,c = 2′-deoxy-2′-fluoro U,C T = thymidine B = inverteddeoxy abasic s = phosphorothioate linkage A = deoxy Adenosine G = deoxyGuanosine G = 2′-O-methyl Guanosine A = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo S/AS “Stab 1” Ribo Ribo — 5 at 5′-end S/AS1 at 3′-end “Stab 2” Ribo Ribo — All Usually AS linkages “Stab 3”2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4” 2′-fluoroRibo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoro Ribo — 1 at 3′-endUsually AS “Stab 6” 2′-O- Ribo 5′ and 3′- — Usually S Methyl ends “Stab7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8” 2′-fluoro2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′ and 3′- — UsuallyS ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11” 2′-fluoro2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′ and 3′-Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS “Stab 14”2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 15” 2′-deoxy2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo 2′-O- 5′ and3′- Usually S Methyl ends “Stab 17” 2′-O- 2′-O- 5′ and 3′- Usually SMethyl Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′- Usually S Methylends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab 20” 2′-fluoro2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS“Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro* 2′-deoxy* 5′and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/AS Methyl* “Stab 26”2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro* 2′-O- 3′-end S/ASMethyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab 29”2′-fluoro* 2′-O- 1 at 3′-end S/AS Methyl* “Stab 30” 2′-fluoro* 2′-O-S/AS Methyl* “Stab 31” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab 32”2′-fluoro 2′-O- S/AS Methyl CAP = any terminal cap, see for example FIG.10. All Stab 00-32 chemistries can comprise 3′-terminal thymidine (TT)residues All Stab 00-32 chemistries typically comprise about 21nucleotides, but can vary as described herein. S = sense strand AS =antisense strand *Stab 23 has a single ribonucleotide adjacent to 3′-CAP*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus *Stab25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus *Stab29, Stab 30, and Stab 31, any purine at first three nucleotide positionsfrom 5′-terminus are ribonucleotides p = phosphorothioate linkage

TABLE V Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methylWait Time*RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL  5 sec 5sec 5 sec N-Methyl 186 233 μL  5 sec 5 sec 5 sec Imidazole TCA 176 2.3mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec  300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL  5 sec 5 sec 5 sec N-Methyl 1245 124 μL  5sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec  300 sec300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* Reagent2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA 2′-O-methyl Wait Time* RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec  S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec  AceticAnhydride 265/265/265 50/50/50 μL 10 sec  10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec  10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec  15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec  30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec  200 sec 200sec  Acetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does notinclude contact time during delivery. Tandem synthesis utilizes doublecoupling of linker molecule

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleicacid molecule comprises a sense strand and a separate antisense strand,each strand having one or more pyrimidine nucleotides and one or morepurine nucleotides; (b) each strand of the nucleic acid molecule isindependently 18 to 27 nucleotides in length; (c) an 18 to 27 nucleotidesequence of the antisense strand is complementary to a human PlateletDerived Growth Factor Receptor (PDGFr) RNA sequence comprising SEQ IDNO: 749; (d) an 18 to 27 nucleotide sequence of the sense strand iscomplementary to the antisense strand and comprises an 18 to 27nucleotide sequence of the human PDGFr RNA sequence; and (e) 50 percentor more of the nucleotides in at least one strand comprise a 2-sugarmodification, wherein the 2′-sugar modification of any of the pyrimidinenucleotides differs from the 2′-sugar modification of any of the purinenucleotides.
 2. The nucleic acid molecule of claim 1, wherein 50 percentor more of the nucleotides in each strand comprise a 2′-sugarmodification.
 3. The nucleic acid molecule of claim 1, wherein the2′-sugar modification is selected from the group consisting of2′-deoxy-2′-fluoro, 2′-O-methyl, and 2′-deoxy.
 4. The nucleic acid ofclaim 3, wherein the 2′-deoxy-2′-fluoro sugar modification is apyrimidine modification.
 5. The nucleic acid of claim 3, wherein the2′-deoxy sugar modification is a pyrimidine modification.
 6. The nucleicacid of claim 3, wherein the 2′-O-methyl sugar modification is apyrimidine modification.
 7. The nucleic acid molecule of claim 4,wherein said pyrimidine modification is in the sense strand, theantisense strand, or both the sense strand and antisense strand.
 8. Thenucleic acid molecule of claim 6, wherein said pyrimidine modificationis in the sense strand, the antisense strand, or both the sense strandand antisense strand.
 9. The nucleic acid molecule of claim 3, whereinthe 2′-deoxy sugar modification is a purine modification.
 10. Thenucleic acid molecule of claim 3, wherein the 2′-O-methyl sugarmodification is a purine modification.
 11. The nucleic acid molecule ofclaim 9, wherein the purine modification is in the sense strand.
 12. Thenucleic acid molecule of claim 10, wherein the purine modification is inthe antisense strand.
 13. The nucleic acid molecule of claim 1, whereinthe nucleic acid molecule comprises ribonucleotides.
 14. The nucleicacid molecule of claim 1, wherein the sense strand includes a terminalcap moiety at the 5′-end, the 3′-end, or both of the 5′- and 3′-ends.15. The nucleic acid molecule of claim 14, wherein the terminal capmoiety is an inverted deoxy abasic moiety.
 16. The nucleic acid moleculeof claim 1, wherein said nucleic acid molecule includes one or morephosphorothioate internucleotide linkages.
 17. The nucleic acid moleculeof claim 16, wherein one of the phosphorothioate internucleotidelinkages is at the 3′-end of the antisense strand.
 18. The nucleic acidmolecule of claim 1, wherein the 5′-end of the antisense strand includesa terminal phosphate group.
 19. The nucleic acid molecule of claim 1,wherein the sense strand, the antisense strand, or both the sense strandand the antisense strand include a 3′-overhang.
 20. A compositioncomprising the nucleic acid molecule of claim 1, in a pharmaceuticallyacceptable carrier or diluent.